CN113069435A - Amomum Hainan polysaccharide PLGA nanoparticles and preparation method thereof - Google Patents
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Abstract
The invention discloses a Hainan villous Amomum fruit polysaccharide PLGA nano particle and a preparation method thereof, wherein the preparation method comprises the following steps: (1) mixing the Hainan fructus amomi polysaccharide solution with the acetone solution of PLGA, carrying out ultrasonic treatment to form primary emulsion, then pouring the primary emulsion into the poloxamer solution, and carrying out ultrasonic treatment to homogenize the primary emulsion; (2) and (2) performing rotary evaporation on the solution obtained in the step (1) to remove acetone, centrifuging to collect supernatant, and freeze-drying the supernatant to obtain the Hainan fructus amomi polysaccharide PLGA nanoparticles. The preparation method of the nano-particle is simple, and compared with the raw material medicine (Hainan fructus amomi polysaccharide), the Hainan fructus amomi polysaccharide PLGA nano-particle prepared by the method has good slow release effect.
Description
Technical Field
The invention belongs to the technical field of nano materials, and relates to a Hainan fructus amomi polysaccharide PLGA nano particle and a preparation method thereof.
Background
Hainan villous Amomum is mainly produced in Chengmei and Lingshui counties, and the history of use is as long as 1300 years. The Hainan villous Amomum fruit not only has the functions of warming and invigorating spleen, eliminating dampness and purging, preventing abortion, treating inflammatory bowel disease, easing pain and resisting inflammation clinically, but also can be used as a food and has the effects of enhancing appetite, relieving fatigue, nourishing organisms and the like. The Hainan fructus amomi polysaccharide is an important active ingredient extracted and separated from Hainan fructus amomi, but has the defects of short action time, low bioavailability and the like due to belonging to macromolecular substances.
The medicament encapsulated by the nanoparticles can prolong the circulation time and improve the bioavailability through the dissolution efficiency. Polylactic-co-glycolic acid (PLGA) has the advantages of good biocompatibility and controllable degradation speed, and the degradation products are lactic acid and glycolic acid, which are one of the byproducts of the body metabolism. Therefore, when it is applied to medicines and biomaterials, it does not produce toxic or side effects, and therefore, it is often made into artificial catheters, drug sustained-release carriers, tissue scaffold materials, etc.
Disclosure of Invention
In order to overcome the defects of fast metabolism in vivo and the like of the Hainan fructus amomi polysaccharide, the invention aims to provide a preparation method of the Hainan fructus amomi polysaccharide PLGA nanoparticles, which has simple preparation process, smaller particle size and good slow release effect.
In order to achieve the technical purpose, the invention adopts the following technical scheme:
the invention provides a preparation method of a Hainan fructus amomi polysaccharide PLGA nano particle, which comprises the following steps:
(1) mixing the Hainan fructus Amomi polysaccharide solution with PLGA acetone solution, performing ultrasonic treatment to form colostrum, pouring the colostrum into poloxamer solution, and performing ultrasonic treatment to homogenize.
(2) And (2) performing rotary evaporation on the solution obtained in the step (1) to remove acetone, centrifuging to collect supernatant, and freeze-drying the supernatant to obtain the Hainan fructus amomi polysaccharide PLGA nanoparticles.
Preferably, in the step (1), the volume ratio of the Hainan fructus amomi polysaccharide solution to the PLGA acetone solution is 1:7.
Preferably, in the step (1), the concentration of the amomum villosum polysaccharides in the amomum villosum polysaccharide solution is 20 mg/mL.
Preferably, in the step (1), the mass ratio of lactic acid to glycolic acid in the PLGA is 75: 25.
Preferably, in the step (1), the concentration of PLGA in the acetone solution of PLGA is 30 mg/mL.
Preferably, in step (1), the volume ratio of the colostrum to the poloxamer solution is 1:7.
Preferably, in the step (1), the mass concentration of the poloxamer solution is 0.7%.
Preferably, in step (1), the ultrasonic treatment is 130W for 2 minutes, and the ultrasonic probe is switched on for 2s and switched off for 3 s.
Preferably, in the step (2), the rotary evaporation condition is 55 ℃ and 30 min.
Preferably, in step (2), 3500rpm is centrifuged for 10 min.
The invention also provides the Hainan villous amomum fruit polysaccharide PLGA nano-particles prepared by the preparation method.
The patent investigates important influence factors in the Hainan fructus amomi polysaccharide nanoparticle preparation process, screens out factors having obvious influence on the encapsulation efficiency of the Hainan fructus amomi polysaccharide nanoparticles and determines the horizontal range through a single-factor test result, then performs test Design by using a response surface Box-Behnken Design test model, analyzes data and obtains the optimal condition combination for preparing the Hainan fructus amomi polysaccharide nanoparticles.
The invention has the beneficial effects that:
the invention uses a double emulsification-solvent volatilization method to prepare the Hainan fructus amomi polysaccharide PLGA nanoparticles, uses the Hainan fructus amomi polysaccharide as a main drug, uses PLGA and poloxamer to encapsulate the Hainan fructus amomi polysaccharide into spherical nanoparticles, and uses a response surface method to optimize the preparation process of the Hainan fructus amomi polysaccharide PLGA nanoparticles, so that the optimized nanoparticles have higher encapsulation efficiency and smaller particle size, and the slow release effect is improved.
Drawings
FIG. 1 shows the results of a one-factor test of an embodiment of the present invention.
FIG. 2 is a 3D and 2D representation of a response surface of an embodiment of the present invention.
FIG. 3 shows the surface states of nanoparticles under a field emission electron microscope, wherein A and B are the forms of the fructus Amomi polysaccharide nanoparticles under different magnifications; c and D are the forms of the blank nanoparticles under different magnifications.
FIG. 4 in vitro release profiles of ALP and ALPP according to an embodiment of the present invention.
Detailed Description
In order to facilitate understanding of the present invention, the technical solutions of the present invention will be further described below with reference to specific embodiments and accompanying drawings, and it should be understood that the embodiments are not intended to limit the present invention.
Examples
Reagent and instrument
1.1 materials and reagents
Fructus Amomi purchased from delirium Green Fu fresh agricultural development Co., Ltd; polylactic-co-glycolic acid (PLGA) was purchased from Shanghai-derived leaf Biotech, Inc.; poloxamer (Poloxamer188, F68) was purchased from Sorba technologies, Inc., Beijing; sephadex G-50, Tween-80, Sephadex G-100 were purchased from Beijing Boototta technologies, Inc.
1.2 instruments and devices
JY92-IIDN ultrasonic cell disruptor, Ningbo Xinzhi Biotech GmbH; 721N spectrophotometer, shanghai instrument electric analyzer ltd; r210 rotary evaporator, BUCHI, switzerland; ThermoscientificVerios G4 UC field emission scanning electron microscope, Thermo Fisher Scientific, USA; ZS90 laser particle size analyzer, Malvern Instrument, uk; HX-10-50B experimental freeze dryer, Shanghai province industries Ltd; AR223CN electronic balance, ohauss instruments (chang) ltd; 5811 high speed refrigerated centrifuge, Eppendorf, Germany.
Extraction of fructus amomi polysaccharide
Completely soaking 500g of Hainan fructus Amomi powder in 6000mL of deionized water, boiling with strong fire, decocting with slow fire for 1 hour, filtering with gauze, decocting the filter residue with distilled water twice, combining the three filtrates, concentrating the filtrate to 500mL, centrifuging at 4000rpm for 10min, and collecting the supernatant. Adding 95% ethanol until ethanol concentration is 80% (v/v), collecting precipitate, and oven drying to obtain crude polysaccharide.
0.1mg/mL of crude Saran polysaccharide solution was added with 4 volumes of Sevage reagent (chloroform: n-butanol (volume ratio 4: 1), magnetically stirred for 20min, centrifuged at 2500rpm for 10min, the supernatant was collected, repeated 5 times, and the supernatant was concentrated, and then the sample was purified by passing through DEAE-52 cellulose column (2.6 cm. times.30 cm) and Sephadex G-100 column (2.6 cm. times.90 cm), and lyophilized to obtain the polysaccharide.
Preparation of semen amomi Hainan polysaccharide PLGA nanoparticles (ALPP)
The internal aqueous phase (10mg of Hainan Amomum villosum polysaccharide dissolved in 0.5mL of ultrapure water to form a Hainan Amomum villosum polysaccharide solution) was mixed with the organic phase (135mg of PLGA solid dissolved in 4.5mL of acetone) and sonicated with an ultrasonic cell disruptor at 130W for 2 minutes (2 s on and 3s off) to form colostrum (water-in-oil). Then 5mL of colostrum was poured into the external aqueous phase (350mg poloxamer188 (F68) dissolved in 50mL of ultra pure water) and homogenized by treatment with an ultrasonic probe 150W for 2 minutes (2 seconds on and 3 seconds off). The acetone was removed by rotary evaporation using a rotary evaporator at 55 ℃ for 30 minutes. The supernatant was then collected by centrifugation at 3500rpm for 10 minutes. The supernatant was then freeze dried and stored sealed.
Fourthly, optimizing and detecting preparation process
4.1 determination of the encapsulation efficiency
Sephadex G-50 was completely soaked in 0.9% NaCl solution for at least 6 hours, and was sufficiently swollen. The plunger of the 2mL syringe was removed and a Whatman filter paper was inserted into the syringe. The Sephadex G-50 gel was carefully added to a syringe, which was then placed into a centrifuge tube to form a microcolumn centrifuge module, and centrifuged (1600rpm, 5 minutes) to remove excess water and compact it for use. Next, 0.5mL of ALPP solution was carefully dropped on top of the gel column and centrifuged at 1600rpm for 5 minutes; continuously adding 1mL of ultrapure water at the top end of the gel column, and centrifuging at 1600rpm for 5 minutes; repeat 2 times. The eluate was made up to 6mL with ultrapure water, and the content of ALP in the eluate was measured by the phenol-sulfuric acid method.
Envelope rate EE% ═ CE/CT × 100% (CE is the content of encapsulated ALP, CT is the total ALP content) 4.2 one-factor experimental design
Selecting 4 factors of the volume ratio of the inner water phase to the oil phase (W1: O), the volume ratio of the colostrum to the outer water phase (colostrum: W2), the mass concentration of F68, the concentration of ALP and the like to carry out single-factor tests so as to screen out the factors which can obviously influence the ALPP encapsulation rate and determine the horizontal range of the factors. Setting the power of the ultrasonic colostrum to be 130W, turning on the ultrasonic for 2s and turning off the ultrasonic for 3s and setting the total time length to be 2min for unifying the test conditions; during ultrasonic multiple emulsion, the power is 150W, the ultrasonic is turned on for 2s and turned off for 3s for 2min, when organic solvent is removed by rotary evaporation to carry out nanoparticle solidification, the rotating speed is 50rpm, the rotary evaporation time is 30min, and the water bath temperature is 55 ℃.
The results show that, in the case of otherwise unchanged conditions, the effect of different volume ratios of internal aqueous phase and oil on the ALPP encapsulation efficiency is shown in fig. 1A, where the encapsulation efficiency increases with increasing oil phase volume, reaching a maximum encapsulation efficiency at 1:6 and beginning to decrease at 1: 8; the encapsulation efficiency gradually decreases with increasing oil phase. The influence of different ALP concentrations on the ALPP encapsulation efficiency is shown in fig. 1B, and it can be seen from the graph that the encapsulation efficiency is substantially constant with the increasing ALPP concentration, so that the factor is not used as a key factor to perform response surface optimization scheme design. The effect of different PE: W2 on ALPP encapsulation efficiency is shown in FIG. 1C, which shows that the encapsulation efficiency increases with the increase of the external aqueous phase volume, reaches a maximum value at 1:8, and slightly decreases at 1: 10. The effect of different F68 concentrations on the packing fraction of ALPPs without changing other conditions is shown in fig. 1D, where the packing fraction increases with increasing F68 concentration, reaching a maximum at a mass concentration of 0.7%, and begins to decrease at 0.9%.
4.3 response surface Experimental design
The Response Surface Method (RSM) is a statistical method which is characterized in that experiments are completed according to reasonable experimental design, data are obtained, a function relation between a response value and an influence factor is calculated, a multiple quadratic regression equation is obtained through fitting, and the optimal response value is found out through the regression equation.
The application takes a single-factor test as a basis, and 3 factors which have more remarkable influence on the ALPP encapsulation rate are screened out as follows: the volume ratio of the inner aqueous phase to the oil phase (A), the volume ratio of the colostrum to the outer aqueous phase (B), and the mass concentration of F68 (C). Experiments were set up with the factors and levels of table 1, the encapsulation efficiency as the response value, the volume ratio of the inner aqueous phase to the oil phase (W1: O), the volume ratio of the colostrum to the outer aqueous phase (PE: W2), and the mass concentration of F68, these 3 factors being independent variables, and the encapsulation efficiency as a dependent variable.
TABLE 1 analysis of the response surface of the factors and levels for the preparation of nanoparticles of fructus Amomi polysaccharide
Based on a single-factor test, 3 factors which have obvious influence on the ALPP encapsulation efficiency are screened out, and the factors are respectively as follows: volume ratio of internal aqueous phase to oil phase (X)1) Volume ratio of colostrum to external aqueous phase (X)2) And mass concentration of F68 (X)3). Responsive to the encapsulation efficiency of ALPP, X1、X2、X3Three factors are independent variables, and Design-Expert10.0.7 response surface software is adopted for experimental Design. A Box-Behnken test design model is selected, the entrapment rate of ALPP is used as a response value, a quadratic regression orthogonal combination test of 17 tests with 3 factors and 3 levels is designed, and the test design and the result are shown in Table 2.
TABLE 2 response surface test results
As shown in Table 2, A, B, C represent X respectively1、X2、X3The encapsulation efficiency varied from 58.91% to 84.90%. Sounding the test resultAfter secondary regression fitting, a fitting equation for obtaining the relationship between the encapsulation efficiency (Y) and the influencing factor X is obtained by surface analysis as follows: y is 82.29-2.27X1-3.15X2+0.83X3+0.51X1X2-1.08X1X3-4.56X2X3-7.86X1 2-3.90X2 2-12.62X3 2
TABLE 3 encapsulation efficiency regression equation coefficient significance test results
According to Table 3, a model P established by characteristic indexes can be obtained<0.05, and the simulation losing item is not significant, which shows that the obtained regression model has higher reliability and can be used for predicting the fructus amomi polysaccharide nanoparticle encapsulation efficiency test. Determining the coefficient R20.9539, correction decision coefficient RAdj 20.9529. The results show that the model has better fitting degree.
The interaction among the parameters can be intuitively reflected by the response surface graph, and the more obvious the paraboloid of the surface graph is, the stronger the interaction among the parameters is, and the weaker the interaction is otherwise. Fig. 2 is a response graph and a contour plot of the encapsulation efficiency of the Hainan fructus amomi polysaccharide PLGA nanoparticles and three factors, which visually reflects the influence of three single-factor variable interaction on the Hainan fructus amomi polysaccharide PLGA nanoparticles Y. The steeper the surface of the three-dimensional response graph is, the more obvious the single-factor interaction is, and the more obvious the contour lines tend to be in an oval shape, so that the single-factor interaction is. Meanwhile, the magnitude of the F value directly reflects the closeness degree of the relationship between each factor and the experimental result, namely: the volume ratio of the inner aqueous phase to the oil phase (W1: O) > the volume ratio of the colostrum to the outer aqueous phase (PE: W2) > the mass concentration solvent concentration of F68.
The Design-Expert10.0.7 software is used for optimizing experimental parameters, and the optimal conditions are as follows: the volume ratio of the inner water phase to the oil phase (W1: O) is 1:7.225, the volume ratio of the colostrum to the outer water phase (PE: W2) is 1:7.179, the concentration of the mass concentration of F68 is 0.716%, and the highest encapsulation rate of the Hainan amomum villosum polysaccharide is 78.547%.
The optimal process conditions obtained by prediction are optimized to obtain: the volume ratio of the inner aqueous phase to the oil phase (W1: O) was 1:7, the volume ratio of the colostrum to the outer aqueous phase (colostrum: W2) was 1:7, and the concentration of the mass concentration of F68 was 0.7%.
4.4 best Process verification
ALPP was prepared according to the optimum process conditions of the response surface, and encapsulation efficiency was measured.
The encapsulation rate of the Hainan fructus amomi polysaccharide nanoparticles is detected to be 79.88 +/-0.47%, and the difference is not large from the predicted value 78.547%, which indicates that the model fitting degree is better.
4.5 analysis of the nanoparticle Properties
The characterized morphology of the nanoparticles was observed using a Field Emission Scanning Electron Microscope (FESEM). The particle size and Zeta potential of nanoparticles (0.01g/L) were measured with a laser particle size analyzer in triplicate. The electron micrograph of the nanoparticles is shown in FIG. 3.
The particle size and potential results of the nanoparticles are shown in table 4, and the particle size of ALPP and BP were not significantly different and were at the same level. However, the absolute value of Zeta potential of ALPP is significantly larger than that of BP, which indicates that the nanoparticle stability of ALPP is better.
TABLE 4 particle size and Zeta potential of the nanoparticles
Note: p < 0.05 compared to BP
4.6 in vitro Release Rate
5mL of ALP and ALPP (1mg/mL) were placed in a dialysis bag and immersed in 50.0mL of PBS (pH 7.4) solution containing 4% Tween-80. Then it was placed in a shaker and shaken horizontally (80rpm) at 37 ℃. At 0.5, 1, 2, 4, 6, 8, 12, 24, 36, 48, 72 and 96h, respectively, 2mL of dialysate was collected while making up to 50mL of dialysate. The collected culture broth was measured for the content of released ALP by the phenol-sulfuric acid method.
The ratio of the amount of ALP released at each time point to the total amount of ALP was the cumulative release rate of ALPP at that time point.
The in vitro release rate of ALPP nanoparticles is shown in fig. 4. The drug release of ALP reached 11.66% in 0.5h, and 58.13% in 72 h. At 0.5h, the fructus amomi polysaccharide is released from ALPP by only 4.5 percent, and the cumulative release amount at 72h is only 38.50 percent. This indicates that ALPP has a better sustained-release function than ALP of the same concentration.
Claims (10)
1. A preparation method of a Hainan fructus amomi polysaccharide PLGA nanoparticle comprises the following steps:
(1) mixing the Hainan fructus amomi polysaccharide solution with the acetone solution of PLGA, carrying out ultrasonic treatment to form primary emulsion, then pouring the primary emulsion into the poloxamer solution, and carrying out ultrasonic treatment to homogenize the primary emulsion;
(2) and (2) performing rotary evaporation on the solution obtained in the step (1) to remove acetone, centrifuging to collect supernatant, and freeze-drying the supernatant to obtain the Hainan fructus amomi polysaccharide PLGA nanoparticles.
2. The method for preparing Hainan fructus amomi polysaccharide-PLGA nanoparticles according to claim 1, wherein in the step (1), the volume ratio of the Hainan fructus amomi polysaccharide solution to the PLGA acetone solution is 1:7.
3. The method for preparing Hainan fructus Amomi polysaccharide-PLGA nanoparticles as claimed in claim 1, wherein in step (1), the concentration of Hainan fructus Amomi polysaccharide in the Hainan fructus Amomi polysaccharide solution is 20 mg/mL.
4. The method for preparing Hainan fructus amomi polysaccharide-PLGA nanoparticles according to claim 1, wherein in the step (1), the mass ratio of lactic acid to glycolic acid in PLGA is 75: 25.
5. The method for preparing Hainan fructus Amomi polysaccharide-PLGA nanoparticles as claimed in claim 1, wherein in step (1), the concentration of PLGA in the acetone solution of PLGA is 30 mg/mL.
6. The method for preparing Hainan fructus Amomi polysaccharide-PLGA nanoparticles as claimed in claim 1, wherein in step (1), the volume ratio of the colostrum to the poloxamer solution is 1:7.
7. The method for preparing Hainan fructus amomi polysaccharide-PLGA nanoparticles according to claim 1, wherein in the step (1), the mass concentration of the poloxamer solution is 0.7%.
8. The method for preparing the fructus amomi Hainan polysaccharide-PLGA nanoparticles according to claim 1, wherein in the step (1), the ultrasonic treatment is 130W for 2 minutes, and the ultrasonic probe is turned on for 2s and turned off for 3 s.
9. The method for preparing Hainan fructus amomi polysaccharide-PLGA nanoparticles according to claim 1, wherein in the step (2), the rotary evaporation condition is 55 ℃, 30 min; centrifuge at 3500rpm for 10 min.
10. A Hainan villous Amomum polysaccharide PLGA nanoparticle, which is prepared by the preparation method of the Hainan villous Amomum polysaccharide PLGA nanoparticle of any one of claims 1 to 9.
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| CN115778921A (en) * | 2022-12-29 | 2023-03-14 | 浙江工业大学 | Dictyophora rubrovalvata polysaccharide-PLGA nanoparticles as well as preparation method and application thereof |
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Application publication date: 20210706 |