CN116035209A - Chitosan modified anthocyanin nanoliposome based on microfluidic technology and application thereof - Google Patents

Chitosan modified anthocyanin nanoliposome based on microfluidic technology and application thereof Download PDF

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CN116035209A
CN116035209A CN202310322983.XA CN202310322983A CN116035209A CN 116035209 A CN116035209 A CN 116035209A CN 202310322983 A CN202310322983 A CN 202310322983A CN 116035209 A CN116035209 A CN 116035209A
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anthocyanin
chitosan
solution
nanoliposome
liposome
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许文涛
朱龙佼
刘海燕
茹苹
兰欣悦
陈可仁
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China Agricultural University
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Abstract

The invention provides a chitosan modified anthocyanin nano liposome based on a microfluidic technology and application thereof. The nano liposome with high encapsulation efficiency (more than 70%) for wrapping anthocyanin is successfully prepared by carrying out systematic evaluation by taking the particle size and the encapsulation efficiency as response values through the optimized control of the ratio of the content of the microfluidic multiphase channel, the buffer solution condition, the flow rate and other coefficients; meanwhile, the introduction of chitosan modification furthest improves the storage time length, temperature resistance, stability and functionality of the nano liposome under a gastrointestinal system, overcomes the difficulty that anthocyanin is difficult to be taken into a functional food due to environmental sensitivity and is used by a human body, reduces the production cost on the basis of improving the bioavailability of anthocyanin, and is beneficial to the development of anthocyanin in the fields of dietary supplements, pharmaceutical industry, cosmetics and the like.

Description

Chitosan modified anthocyanin nanoliposome based on microfluidic technology and application thereof
Technical Field
The invention belongs to the fields of molecular biological engineering technology and anthocyanin application, and relates to a method design of a microfluidic technology in anthocyanin liposome preparation and food nutrition application of anthocyanin exemplified by Aronia melanocarpa.
Background
Anthocyanin is used as a natural water-soluble pigment and has wide biological activities such as antioxidation, anti-inflammatory, anticancer, anti-obesity and anti-diabetes. However, the compound beverage is very sensitive to temperature, illumination, pH, active oxygen and other condition changes, and particularly after being taken by a human body, the complex internal environment of the human body easily causes the compound beverage to lose the original functional activity and the nutritive value, so that the actual gastrointestinal absorption rate and the bioavailability of the compound beverage are low, and the application of the compound beverage in the food industry is greatly limited.
Nanomaterials are an important discovery for protecting environmentally sensitive bioactive molecules and are also an effective method of drug delivery that can provide a protective barrier to bioactive compounds in a manner that encapsulates them under adverse environmental conditions, thereby better aiding biological applications. Among them, liposomes having phosphate and cholesterol as a skeleton have been widely and ripely used in industries such as food molecular loading and pharmaceutical industry due to their excellent biocompatibility, biodegradability, low toxicity and immunogenicity. However, the liposome also faces the problems of short half-life, easy degradation, aggregation, material oxidation and the like in the gastrointestinal tract, and Chitosan (CS) is taken as a cationic polysaccharide biopolymer, has good mucous membrane adhesion and intestinal penetrating stability, can effectively increase the retention time of biomolecules at absorption sites, and helps the liposome to improve the bioavailability.
In many preparation methods of the liposome, the microfluidic technology has the outstanding advantages of realizing uniform mixing of multiple phases due to the real-time control of material flow rate and proportion, solving the problems of long preparation time, poor uniformity, uncontrollable particle size and the like of the traditional methods such as a film dispersion method, a reverse evaporation method, an organic solvent injection method and the like, and leading the liposome preparation to have potential of industrial production.
Disclosure of Invention
Based on the problem that anthocyanin with good biological activity and function is difficult to absorb and utilize in vivo, and the significant advantages of chitosan in vivo stability and the preparation of liposome by a microfluidic technology are combined, the technical problem to be solved by the invention is as follows: the synthesis of liposome capable of wrapping anthocyanin is optimally controlled by applying a microfluidic technology, and chitosan modification is introduced on the basis, so that an anthocyanin nano liposome delivery system with low particle size, high loading capacity and good stability is constructed, and scientific basis is provided for research and application of novel functional foods.
In one aspect, the invention provides a method for preparing a chitosan-modified anthocyanin nanoliposome delivery system based on a microfluidic technology, comprising the following steps:
(1) Lecithin and cholesterol were mixed at 1: 1-5: 1 in the mass ratio of absolute ethyl alcohol solution, and carrying out ultrasonic treatment until the absolute ethyl alcohol solution is completely dissolved to obtain a lipid ethyl alcohol solution which is used as a lipid phase of microfluidic equipment; and diluting anthocyanin with a PBS buffer solution with the pH value of 6.5-7.2 to obtain the water phase of the microfluidic device, wherein the concentration of the diluted anthocyanin is 0.1-0.3 mg/mL. Preferably, the mass ratio of lecithin to cholesterol is controlled to be 5:1, as a lipid phase, aronia melanocarpa (Aronia melanocarpa, AM) at a concentration of 0.2 mg/mL was dissolved in PBS (ph=6.7), as an aqueous phase;
(2) The aqueous and lipid phases were passed through a micropump at 1: 1-5: 1 and the total flow rate is 1.5-4 mL/min, and the anthocyanin nanoliposome is obtained. The liposome sample container is kept in an open state and kept at a temperature of 4 ℃ for at least 4-8 hours so as to stabilize and remove absolute ethyl alcohol, and is kept for standby at the temperature of 4 ℃. Preferably, the flow rate ratio is controlled at 3:1, total flow rate is 4 mL/min, and after 6 h at 4 ℃, absolute ethyl alcohol is stabilized and removed, and the mixture is preserved for standby at 4 ℃;
(3) And (3) preparing a chitosan solution, dissolving 1.00-g chitosan in 1% glacial acetic acid solution, stirring until the chitosan solution is clear, and diluting the chitosan solution into 0.1-1% chitosan solution. Preferably, the chitosan solution is diluted to 0.7%;
(4) Adding the liposome solution in the step 2 into the chitosan solution in the step 3, and mixing the liposome solution with the chitosan solution in the volume ratio of 1:1, stirring for 0.5-2 h at 45-55 ℃, standing for 0.5-2 h, centrifuging for 20-60 min at 2000-5000 rpm and 4 ℃, removing redundant chitosan, discarding supernatant, adding PBS for resuspension, and storing at 4 ℃ for later use to obtain chitosan modified anthocyanin nanoliposome. Preferably, stirring at 50deg.C for 1 h, standing for 0.5 h, centrifuging at 3000 rpm at 4deg.C for 30 min, removing excessive chitosan, discarding supernatant, adding PBS, and storing at 4deg.C.
On the other hand, the anthocyanin nanoliposome prepared by the preparation method and the application thereof in drug delivery, targeted therapy or path verification research are related.
The invention has the technical effects that:
(1) The anthocyanin nanoliposome is prepared by the microfluidic technology for the first time, so that the problems of long preparation time, poor uniformity, larger particle size and the like of the nanoliposome prepared by the traditional method are solved, and the nanoliposome has better mass industrialized production potential.
(2) Taking the Aronia melanocarpa anthocyanin with the most abundant content in the natural plants as an example for the first time, as a functional molecule for embedding and delivering, the method not only overcomes the problem of anthocyanin environmental sensitivity, improves the bioavailability of anthocyanin, but also reduces the production cost to the greatest extent in the aspect of raw material introduction.
(3) The modifier is used for modifying the Aronia melanocarpa anthocyanin nanoliposome by chitosan, so that the overall stability and functionality of the Aronia melanocarpa anthocyanin nanoliposome are improved, the stay time of anthocyanin in the body is prolonged, the anthocyanin can better exert the physiological activity in the body, and the bioavailability is improved.
Drawings
FIG. 1 is a graph showing the effect of two-phase flow on particle size distribution.
Figure 2 effect of total flow rate on particle size distribution.
Figure 3 effect of anthocyanin concentration on encapsulation efficiency.
FIG. 4 effect of lipid-bile ratio on encapsulation efficiency.
FIG. 5 effect of pH of PBS on encapsulation efficiency.
FIG. 6 effect of anthocyanin concentration versus lipid-to-bile ratio, anthocyanin concentration versus pH, lipid-to-bile ratio versus pH interaction on encapsulation efficiency. (a) anthocyanin concentration interacts with lipid-to-bile ratio; (B) anthocyanin concentration interacts with pH; (C) interaction of lipid-bile ratio with pH.
FIG. 7 characterization of anthocyanin nanoliposomes. (A) particle size map; (B) Potential diagram
FIG. 8 effect of chitosan concentration on average particle size and potential.
FIG. 9 characterization of anthocyanin nanoliposomes after chitosan modification. (A) particle size map; (B) potential diagram.
FIG. 10 effect of storage time on anthocyanin nanoliposome particle size. (a) before ultrasound; (B) after ultrasound.
FIG. 11 effect of temperature on anthocyanin nanoliposome stability.
Figure 12 simulates the effect of intestinal juice on anthocyanin nanoliposome stability. (a) simulating intestinal juice; (B) simulation of gastric juice.
Detailed Description
The invention discloses a development method of a chitosan modified anthocyanin nanoliposome delivery system based on a microfluidic technology, and a person skilled in the art can refer to the content of the invention to properly improve the technological parameters. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the methods and applications described herein, and in the practice and application of the techniques of this invention, without departing from the spirit or scope of the invention.
Example 1 preparation and optimization of Aronia melanocarpa anthocyanin-coated liposomes based on microfluidic platform
AM with the most abundant anthocyanin content in the natural plants is used as a functional molecule to carry out liposome embedding delivery, and the product is called AM-Lip for short. Considering that the size of the nano-carrier is closely related to the gastrointestinal tract absorption effect and the transportation and removal efficiency in body fluid, and corresponds to the operation level of the micro-fluidic technology, the main judgment standard is to ensure that the particle size and encapsulation efficiency of the constructed liposome are optimal, so the following optimization is carried out in research:
(1) Optimization of flow rate of aqueous phase and lipid phase
AM at a concentration of 0.2 mg/mL was dissolved in PBS (ph=6.7) to give an aqueous phase; the mass ratio of lecithin to cholesterol is controlled to be 5:1, the lipid phase was used to control the flow rate of the aqueous phase and the lipid phase in which anthocyanin was dissolved at a flow rate gradient ratio of 1 to 3, and the effect on the liposome particle size and the polydispersity index (PDI) was examined. As shown in fig. 1, the liposome size gradually decreases (422.8 to 118.4 nm) from a low flow rate ratio (1:1) to a high flow rate ratio (3:1). Since the PDI value at 0.3 is considered to be monodisperse, i.e. better uniformity, the two-phase flow ratio 3 is chosen: 1 follow-up study was performed.
(2) Total flow ratio optimization
AM at a concentration of 0.2 mg/mL was dissolved in PBS (ph=6.7) to give an aqueous phase; the mass ratio of lecithin to cholesterol is controlled to be 5:1, setting into a lipid phase; the effect of the flow rate gradient ratio of 1.5 to 4 on the liposome particle size and PDI was investigated by controlling the flow rate of aqueous phase and lipid phase of dissolved anthocyanin. As shown in FIG. 2, the particle size and PDI coefficient decreased with the increase of the total flow rate, so that it was confirmed that the flow rate was controlled at 4 mL/min, and the subsequent study was conducted.
(3) AM embedding concentration optimization
The flow rate ratio was controlled at 3:1, total flow rate 4 mL/min, mass ratio of lecithin to cholesterol was controlled to 5:1, setting into a lipid phase; the effect of anthocyanin concentration on liposome encapsulation efficiency was probed with 0.1 to 0.3 anthocyanin dissolved in PBS (ph=6.7) as the aqueous phase. According to the pH differential method, the content C1 of free anthocyanin and the total content C2 of anthocyanin are calculated according to the following formula, and the anthocyanin encapsulation efficiency is obtained. Wherein A refers to KCl buffer and CH at pH 1.0 and pH 4.5 3 The difference in maximum absorbance of the COONa buffer diluted samples; MW refers to the molecular weight of AM anthocyanin; DF refers to the dilution factor; epsilon is the extinction coefficient (26900 mol/L cm); l is the path length (cm).
Figure SMS_1
As shown in fig. 3, the encapsulation efficiency of the liposomes increased with increasing AM concentration until the encapsulation efficiency of the liposomes began to decrease after the concentration exceeded 0.2 mg/mL, suggesting that the concentration was beyond the saturation point at which the stabilized liposomes could be loaded, so the optimal anthocyanin concentration of 0.2 mg/mL was selected for subsequent validation.
(4) Phospholipid and cholesterol ratio optimization
The flow rate ratio was controlled at 3:1, total flow rate 4 mL/min, AM at a concentration of 0.2 mg/mL was dissolved in PBS (ph=6.7), and the aqueous phase was set to give a mass ratio of lecithin to cholesterol of 1:1 to 5:1 dissolving in absolute ethyl alcohol to form a lipid phase; the effect of lipid-to-bile ratio on liposome encapsulation efficiency was investigated. As shown in fig. 4, when the ratio of lecithin to cholesterol is 5: at 1, the encapsulation efficiency reached an optimum of 70.21%, and therefore this ratio was chosen for subsequent investigation.
(5) Optimum pH optimization of aqueous phase
The flow rate ratio was controlled at 3:1, total flow rate 4 mL/min, dissolving AM with concentration of 0.2 mg/mL in PBS with pH of 6.5-7.2, setting the aqueous phase, and controlling mass ratio of lecithin and cholesterol to be 5:1, setting the liposome as a lipid phase, detecting the influence of the pH value of PBS on liposome encapsulation efficiency. As shown in fig. 5, when the pH was near neutral, the encapsulation efficiency of the liposome reached the optimum, suggesting that the liposome lipid phase components were kept stable without hydrolysis to the maximum at this pH, and thus the pH was selected for subsequent studies.
(6) Response surface optimization
On the basis of single-factor optimization of (3), 4 and 5), anthocyanin concentration (A), lipid-cholesterol ratio (B) and pH value (C) are selected as investigation factors, encapsulation efficiency is taken as a response value, a Box-Benhnken method is adopted to carry out response surface experimental design on liposome preparation experiments, and experimental factors and codes are shown in table 1.
TABLE 1 response surface factor level Table
Figure SMS_2
The experimental set was specifically set up to determine the optimal process parameters by performing regression analysis in response to the surface analysis model (table 2).
Table 2 response surface combinations design and experimental results for encapsulation efficiency
Figure SMS_3
Finally, constructing and obtaining a quadratic polynomial regression equation with the lipid-to-bile ratio (A), the AM concentration (B) and the pH value (C) as independent variables and the encapsulation efficiency (Y) as a response dependent variable: y= 70.13+2.70 A+7.75 B+2.03 C+2.41 AB+0.46 AC+0.87 BC-18.80A 2 -13.54 B 2 -18.02 C 2 （R 2 = 0.9938) and a response surface pattern (fig. 6), according to the software analysis result, the optimal AM-Lip preparation process conditions are obtained: AM concentration 0.2 mg/mL (FIG. 6A), lipid-to-bile ratio 5: at a pH of 6.7 (FIG. 6C) of 1 mg/mg (FIG. 6B), the theoretical optimal encapsulation efficiency was 71.44%. Meanwhile, the average particle diameter of CS-AM-Lip is measured by a Markov particle diameter instrument(FIG. 7A) and potential (FIG. 7B).
Example 2 preparation and optimization of chitosan-modified anthocyanin liposome delivery platform
Preparing AM-Lip according to the optimized conditions of the embodiment 1, dropwise adding the same volume of the AM-Lip into 0.1% -1% chitosan solution, stirring at 50 ℃ for 1. 1 h, standing for 0.5 h, centrifuging at 3000 rpm and 4 ℃ for 30 min, removing redundant chitosan, discarding supernatant, adding PBS, and resuspension to obtain chitosan modified anthocyanin nanoliposome (CS-AM-Lip), and exploring the influence of chitosan solutions with different concentrations on the particle size and potential of the anthocyanin nanoliposome. As shown in FIG. 8, the particle size of the liposome gradually increased with increasing chitosan concentration, and particularly at a concentration of 0.70%, the Zeta potential reached a maximum value, and then the potential slightly decreased with increasing concentration, suggesting that the binding site on the surface of the liposome for CS had reached saturation, so that AM-Lip was selectively modified with 0.70% chitosan concentration, and the average particle size (FIG. 9A) and potential (FIG. 9B) of CS-AM-Lip were measured by a Markov particle size meter, and it was found that the particle size slightly increased after modifying chitosan.
Example 3 stability validation of chitosan-modified anthocyanin liposome delivery platform
Finally, the present study was based on the resulting CS-AM-Lip, and its stability was systematically analyzed for better assessment of its subsequent practical application potential.
(1) Time stability
The AM-Lip and CS-AM-Lip solutions were stored at 4deg.C and the particle size change of anthocyanin nanoliposomes was measured at different time nodes (0, 2, 5, 10, 15, 20, 25, 30 d), respectively. As a result, as shown in fig. 10, the material particle size change tended to slightly rise with the increase in storage time, but tended to be stable as a whole. Meanwhile, compared with AM-Lip, the nano material modified by chitosan can effectively improve the oxidization condition of liposome and effectively protect anthocyanin, but because the chitosan has adhesiveness, a small amount of chitosan can be adhered together in the storage process, so that the particle size is enlarged (figure 10A), and the nano material can be dispersed again after ultrasound (figure 10B).
(2) Thermal stability
Heating AM-Lip and CS-AM-Lip solutions at different temperatures (4deg.C, 20deg.C, 40deg.C, 60deg.C, 80deg.C) for 30 min, respectively, according to formula
Figure SMS_4
Wherein EEt is encapsulation efficiency over a period of time, EE0 is original encapsulation efficiency, and leakage rate of anthocyanin nanoliposome is measured. As shown in fig. 11, the leakage rate of AM-Lip increases with increasing temperature, suggesting that the increased temperature causes degradation and destruction of the liposome membrane, resulting in destruction of the phospholipid bilayer structure, resulting in anthocyanin leakage from the liposome. The degradation rate of CS-AM-Lip is obviously improved, which shows that the chitosan modification can further reduce the damage effect of high temperature to liposome, and more effectively protect anthocyanin and reduce degradation and loss.
(3) Simulating gastrointestinal fluid stability
The release conditions of the AM-Lip and CS-AM-Lip solutions in gastrointestinal fluids were analyzed by simulating artificial intestinal gastric juice. Experimental study of simulated intestinal fluid: a certain amount of AM-Lip and CS-AM-Lip solutions were mixed with an equal volume of simulated intestinal fluid, placed in a shaking table at 37℃and shaken in the dark at 200 rpm for 5 h, sampled every half hour for 0.5 mL, and the leak rate calculated. The blank nanoliposome served as a control and the test was repeated three times. The results are shown in FIG. 12A, where the leakage rate of AM-Lip is gradually increased in simulated intestinal fluid, indicating a sustained release of anthocyanin. Among them, in 1 h, the leakage rate of AM-Lip is relatively low, and the overall tends to be smooth. After the reaction time is gradually prolonged, the leakage rate is gradually increased until the anthocyanin is gradually released completely. The degradation rate of CS-AM-Lip is obviously reduced, and the coating formed on the surface of the liposome due to chitosan can effectively prevent lipase and bile salt from entering into lipid double layers, so that the stability of the liposome in intestinal tracts is enhanced. As can be seen from fig. 12B, the stability of the nano-liposome in simulated gastric fluid is higher than that of intestinal fluid, because the pH value of gastric fluid is lower, the degradation of the nano-liposome is slower, and the modification of chitosan is also helpful to reduce the damage of enzymes and acid in gastric fluid to the liposome, so that the stability of anthocyanin nano-liposome is improved.
Reagents and apparatus for use in the methods of the present invention are commercially available. The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (10)

1. A preparation method of chitosan modified anthocyanin nanoliposome based on microfluidic technology is characterized by comprising two steps of nanoliposome preparation and anthocyanin modification.
2. The method of claim 1, wherein the step of nanoliposome preparation comprises:
(1) Lecithin and cholesterol were mixed at 1: 1-5: 1 in the mass ratio of absolute ethyl alcohol solution, and carrying out ultrasonic treatment until the absolute ethyl alcohol solution is completely dissolved to obtain a lipid ethyl alcohol solution which is used as a lipid phase of microfluidic equipment; diluting anthocyanin with PBS buffer solution with pH value of 6.5-7.2 to concentration of 0.1-0.3 mg/mL, and taking the anthocyanin as water phase of microfluidic equipment;
(2) The aqueous and lipid phases were passed through a micropump at 1: 1-5: 1 and the total flow rate is 1.5-4 mL/min, and the anthocyanin nanoliposome is obtained. The liposome sample container is kept in an open state and kept at the temperature of 4 ℃ for at least 4-8 hours so as to stabilize and remove the absolute ethyl alcohol, and is kept for standby at the temperature of 4 ℃.
3. The method of preparing according to claim 1, wherein the anthocyanin modification step comprises:
(1) Preparing a chitosan solution, dissolving 1.00-g chitosan in 1% glacial acetic acid solution, stirring until the solution is clear, and diluting the solution into 0.1-1% chitosan solution;
(2) Adding the liposome solution in the step 2 into the chitosan solution in the step 3, and mixing the liposome solution with the chitosan solution in the volume ratio of 1:1, stirring for 0.5-2 h at 45-55 ℃, standing for 0.5-2 h, centrifuging for 20-60 min at 2000-5000 rpm and 4 ℃, removing redundant chitosan, discarding supernatant, adding PBS for resuspension, and storing at 4 ℃ for later use to obtain chitosan modified anthocyanin nanoliposome.
4. The preparation method according to claim 2, wherein the mass ratio of lecithin to cholesterol is controlled to be 5:1 was set as a lipid phase, anthocyanin at a concentration of 0.2 mg/mL was dissolved in PBS (ph=6.7), and set as an aqueous phase.
5. The production method according to claim 2, wherein the flow rate ratio is controlled to 3:1, total flow rate 4 mL/min, and after 6 h storage at 4 ℃ to stabilize and remove absolute ethanol, storage at 4 ℃ for later use.
6. A method of preparation according to claim 3, wherein the chitosan solution is diluted to a concentration of 0.7%.
7. The method according to claim 3, wherein the chitosan modification experimental parameters are: stirring at 50deg.C for 1. 1 h, standing for 0.5 h, centrifuging at 3000 rpm and 4deg.C for 30 min, removing excessive chitosan, discarding supernatant, adding PBS, and storing at 4deg.C.
8. Anthocyanin nanoliposomes prepared by the method of any one of claims 1 to 7.
9. The use of the preparation method according to any one of claims 1 to 7 for preparing liposomes encapsulating different types of anthocyanins or anthocyanins analogues.
10. The anthocyanin nanoliposome prepared by the preparation method according to any one of claims 1-7, which is applied to the fields of drug delivery, targeted therapy and pathway verification.
CN202310322983.XA 2023-03-30 2023-03-30 Chitosan modified anthocyanin nanoliposome based on microfluidic technology and application thereof Pending CN116035209A (en)

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