Hyaluronic acid modified astaxanthin liposome, preparation method and application thereof, and oral medicine for treating CD44 overexpression diseases

Abstract

The invention discloses hyaluronic acid modified astaxanthin liposome, a preparation method and application thereof and an oral medicine for treating CD44 overexpression diseases, and belongs to the technical field of biological materials. According to the technical scheme, hyaluronic acid modified astaxanthin lipidosome is combined with a CD44 receptor through hyaluronic acid, and astaxanthin is delivered to liver cancer cells over-expressed by the CD44 receptor in a targeted mode; according to the hyaluronic acid modified astaxanthin lipidosome, the hyaluronic acid modified lipidosome is used as a carrier of astaxanthin, and the hyaluronic acid modified astaxanthin lipidosome is delivered in an oral administration mode; the liposome comprises lecithin and phytosterol ester; and the hyaluronic acid modified astaxanthin liposome takes lecithin and phytosterol ester as wall materials. The invention is applied to the aspect of CD44 overexpression disease treatment, and solves the problem that the application of astaxanthin in preparation of oral medicines for treating CD44 overexpression diseases is limited due to the fact that astaxanthin cannot be well combined with a targeted biomarker CD44 receptor on liver cancer cells when astaxanthin is orally delivered in the prior art.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological materials, and particularly relates to a hyaluronic acid modified astaxanthin liposome, a preparation method and application thereof, and an oral drug for treating CD44 overexpression diseases. BACKGROUND

[0002] Astaxanthin (AST) belongs to ketone carotenoids, and has a chemical formula of C 40 H 52 O4. It is in a red crystalline state at normal temperature, is insoluble in water, is easily soluble in organic solvents such as chloroform and dichloromethane, and is widely distributed in the form of fatty acid esters in nature. As a natural antioxidant, astaxanthin can effectively resist the damage of free radicals to human cells, and has multiple protective effects on inflammation, oxidative stress, tumors and DNA damage. However, due to the special chemical structure (highly unsaturated molecular structure) of astaxanthin, the stability of astaxanthin is poor, and astaxanthin has strong lipophilicity, which leads to low solubility, thereby limiting the expansion of the application field of astaxanthin.

[0003] In recent years, there have been studies on the use of liposomes, microcapsules, emulsions and solid lipid nanoparticles for delivering astaxanthin to improve solubility and enhance bioavailability. Among them, the liposome has a phospholipid bilayer structure, has good biocompatibility and biodegradability, and can significantly improve the stability and in vivo delivery efficiency of hydrophobic drugs. Cluster of differentiation 44 (CD44) is a transmembrane glycoprotein, and CD44 can be used as a tumor targeting biomarker. Targeted drug delivery technology can accurately deliver therapeutic drugs to a specific site, that is, by using a ligand to bind to CD44 receptors on target cells, the drug concentration at the treatment site is significantly increased, the drug dosage is reduced, and the drug side effects are reduced.

[0004] However, when astaxanthin is delivered orally by the prior art, astaxanthin cannot be well combined with the targeting biomarker CD44 receptor on hepatocarcinoma cells, thereby limiting the application of astaxanthin in the preparation of oral drugs for treating CD44 overexpression diseases. SUMMARY

[0005] In view of the deficiencies of the prior art, the technical problem to be solved by the present application is that when astaxanthin is delivered orally by the prior art, astaxanthin cannot be well combined with the targeting biomarker CD44 receptor on hepatocarcinoma cells, thereby limiting the application of astaxanthin in the preparation of oral drugs for treating CD44 overexpression diseases. The present application provides a hyaluronic acid modified astaxanthin liposome which can be delivered orally, can be well combined with CD44 receptors, has a high hepatocarcinoma cell uptake rate, and has good stability, a preparation method and application thereof, and an oral drug for treating CD44 overexpression diseases.

[0006] To solve the technical problem, the technical scheme adopted by the present application is: The present application provides the use of hyaluronic acid modified astaxanthin liposomes in the preparation of oral drugs for treating CD44 overexpression diseases. The hyaluronic acid modified astaxanthin liposomes use hyaluronic acid modified liposomes as carriers of astaxanthin and are delivered by oral administration. The liposomes comprise lecithin and phytosterol esters, and the hyaluronic acid modified astaxanthin liposomes use lecithin and phytosterol esters as wall materials.

[0007] In some embodiments, the hyaluronic acid is combined with DSPE-PEG 2000 to form DSPE-PEG 2000 -HA; after the astaxanthin is encapsulated by lecithin and phytosterol esters, the astaxanthin is modified by DSPE-PEG 2000 -HA to obtain the hyaluronic acid modified astaxanthin liposomes.

[0008] In some embodiments, the content of DSPE-PEG 2000 -HA in the hyaluronic acid modified astaxanthin liposomes is 285-507 μg / mL.

[0009] In some embodiments, the content of DSPE-PEG 2000 -HA in the hyaluronic acid modified astaxanthin liposomes is 417.20 ± 14.651 μg / mL.

[0010] The present application provides the use of hyaluronic acid modified astaxanthin liposomes in the preparation of oral drugs for treating CD44 overexpression diseases.

[0011] In some embodiments, the mass ratio of lecithin to phytosterol esters is 5-10:1.

[0012] In some embodiments, the particle size of the hyaluronic acid modified astaxanthin liposomes is 130-160 nm, the PDI is less than 0.4, and the Zeta is less than or equal to -27.8 mV.

[0013] The present application also provides a preparation method of the hyaluronic acid modified astaxanthin liposomes according to any one of the above technical solutions, comprising: mixing and dissolving hyaluronic acid, N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide to obtain a uniform solution, and mixing the uniform solution with DSPE-PEG2000 -NH2 mixed, stirred at room temperature, filtered to obtain a viscous solution, the solvent was evaporated, and further precipitated by adding acetone, and the precipitate was dried to obtain DSPE-PEG 2000 -HA.

[0014] In some embodiments, further comprising: adding lecithin and phytosterol ester into chloroform solution, then adding astaxanthin-chloroform stock solution, obtaining a liposome film after evaporation under reduced pressure, adding ultrapure water to completely dissolve the film, and preparing Lip-AST by crushing and extruding; mixing the Lip-AST with the DSPE-PEG 2000 -HA solution uniformly, and obtaining hyaluronic acid modified astaxanthin liposomes by water bath reaction.

[0015] In some embodiments, the encapsulation rate of astaxanthin is greater than or equal to 77%.

[0016] The application also provides an oral drug for treating CD44 overexpression diseases, comprising the hyaluronic acid modified astaxanthin liposomes according to any one of the technical solutions described above.

[0017] Compared with the prior art, the application has the following beneficial effects: The application provides a use of hyaluronic acid modified astaxanthin liposomes in the preparation of an oral drug for treating CD44 overexpression diseases, wherein astaxanthin is encapsulated by using liposomes with a specific composition, thereby effectively improving the biocompatibility and stability of astaxanthin, realizing oral utilization of astaxanthin, and realizing active targeting of the hyaluronic acid modified astaxanthin liposomes to CD44 by cooperating with hyaluronic acid modification, so that astaxanthin is delivered in a high conversion rate, and the treatment effect on CD44 overexpression diseases is realized. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 A synthesis diagram of DSPE-PEG 2000 -HA provided in the embodiments of the application is shown in the figure. Figure 2 A synthesis diagram of HA, DSPE-PEG 2000 -HA provided in the embodiments of the application is shown in the figure. 2000 An infrared spectrum diagram of DSPE-PEG Figure 3 A particle size determination data diagram of 5% HA modified astaxanthin liposomes provided in Embodiment 1 of the application is shown in the figure. Figure 4 A particle size determination data diagram of 10% HA modified astaxanthin liposomes provided in Embodiment 2 of the application is shown in the figure. Figure 5 A particle size determination data diagram of 15% HA modified astaxanthin liposomes provided in Embodiment 3 of the application is shown in the figure. Figure 6 Particle size determination data graph of 20% HA modified astaxanthin liposomes provided in the present application embodiment 4; Figure 7 Transmission electron microscopy graph of 5% HA modified astaxanthin liposomes provided in the present application embodiment 1; Figure 8 Transmission electron microscopy graph of 10% HA modified astaxanthin liposomes provided in the present application embodiment 2; Figure 9 Transmission electron microscopy graph of 15% HA modified astaxanthin liposomes provided in the present application embodiment 3; Figure 10 Transmission electron microscopy graph of 20% HA modified astaxanthin liposomes provided in the present application embodiment 4; Figure 11 HA content graph in hyaluronic acid modified astaxanthin liposomes provided in the present application embodiments 1-4; Figure 12 Astaxanthin encapsulation rate in hyaluronic acid modified astaxanthin liposomes provided in the present application embodiments 1-4; Figure 13 Particle size change graph after storage of hyaluronic acid modified astaxanthin liposomes provided in the present application embodiments 1-4; Figure 14 PDI change graph after storage of hyaluronic acid modified astaxanthin liposomes provided in the present application embodiments 1-4; Figure 15 Zeta potential change graph after storage of hyaluronic acid modified astaxanthin liposomes provided in the present application embodiments 1-4; Figure 16 DPPH free radical scavenging activity graph of hyaluronic acid modified astaxanthin liposomes provided in the present application embodiments 1-4; Figure 17 Conversion value of hyaluronic acid modified astaxanthin liposomes provided in the present application embodiment 3 and free AST of comparative example 1; Figure 18 Influence diagram of hyaluronic acid modified astaxanthin liposomes provided in the present application embodiments 1-4 and free AST on hek-293 cell activity; Figure 19 Cell uptake statistical graph of astaxanthin liposomes modified by different HA proportions in the present application embodiment and free AST; Figure 20 Cell uptake fluorescence microscope shooting graph of astaxanthin liposomes modified by different HA proportions in the present application embodiment and free AST; Figure 21A schematic diagram of the effects of astaxanthin liposomes with different HA ratios in the embodiments of the present application on the viability of huh-7 cells. DETAILED DESCRIPTION

[0019] The technical solutions in the specific embodiments of the present application will be described in detail below. Obviously, the described embodiments are only part of the specific implementations of the general technical solution of the present application, rather than all the implementations. Based on the general concept of the present application, all other embodiments obtained by those of ordinary skill in the art fall within the scope of protection of the present application.

[0020] In one aspect, the present application provides a use of hyaluronic acid modified astaxanthin liposomes in the preparation of an oral drug for treating CD44 overexpression diseases. The hyaluronic acid modified astaxanthin liposomes are combined with CD44 receptors through hyaluronic acid, and target astaxanthin to liver cancer cells with CD44 receptor overexpression. The hyaluronic acid modified astaxanthin liposomes use hyaluronic acid modified liposomes as carriers of astaxanthin, and deliver the hyaluronic acid modified astaxanthin liposomes through oral administration. The liposomes include lecithin and phytosterol esters. The hyaluronic acid modified astaxanthin liposomes use lecithin and phytosterol esters as wall materials.

[0021] The above technical solution uses hyaluronic acid to couple with phospholipid material, uses lecithin and phytosterol esters as wall materials, and prepares a new type of hyaluronic acid (Hyaluronic Acid, HA) modified liposome for delivering astaxanthin (Astaxanthin, AST), so as to improve the stability of astaxanthin, improve the water solubility, increase the oral bioavailability, antioxidant property, and improve the cell targeting performance. The hyaluronic acid can specifically recognize the overexpressed CD44 receptor, target the modified liposome, improve the efficiency and accuracy of astaxanthin delivery, and can be applied to prevent and / or treat diseases related to oxidative stress, inflammation, and CD44 receptor overexpression. The drug containing the hyaluronic acid modified astaxanthin liposome can be administered orally, and the oral availability is improved and the intestinal retention time is prolonged.

[0022] It should be noted that hyaluronic acid is a polysaccharide composed of repeating disaccharide units, in which N-acetylglucosamine and glucuronic acid are connected by alternating β-1, 3 and β-1, 4 bonds. HA has a high affinity for CD44 receptors and can be used as a tumor targeting modification of liposomes. The present application uses HA to couple with phospholipid material, uses lecithin and phytosterol esters as wall materials, and prepares a new type of HA modified liposome for delivering astaxanthin, so as to improve the stability of astaxanthin, improve the water solubility, increase the oral bioavailability, antioxidant property, and improve the cell targeting performance.

[0023] In some embodiments, the hyaluronic acid is coupled with DSPE-PEG 2000amine conjugation to form DSPE-PEG 2000 -HA; astaxanthin is encapsulated by lecithin and phytosterol ester, and then modified by DSPE-PEG 2000 -HA to obtain hyaluronic acid modified astaxanthin liposomes.

[0024] In the technical solution, the content of DSPE-PEG 2000 -HA is dissolved in water, and the above technical solution adopts a post-insertion method to modify HA on the liposomes to realize the construction of HA-liposome-AST complexes.

[0025] In some embodiments, the content of DSPE-PEG 2000 -HA in the hyaluronic acid modified astaxanthin liposomes is 285-507 μg / mL. 2000 -HA in the hyaluronic acid modified astaxanthin liposomes is 417.20 ± 14.651 μg / mL.

[0026] The content of DSPE-PEG 2000 -HA in the hyaluronic acid modified astaxanthin liposomes is 285-507 μg / mL. 2000 The hyaluronic acid modified astaxanthin liposomes with the content of DSPE-PEG 2000 -HA of 285-507 μg / mL correspond to 15% HA@Lip, and after being stored for 30 days, the particle size is reduced, the PDI does not change obviously, and the absolute value of the Zeta potential is greater than 25 mV, so that the stability is the best.

[0027] The content of DSPE-PEG 2000 -HA in the hyaluronic acid modified astaxanthin liposomes is limited, so as to balance the modification coverage and targeting, the drug loading and the drug stability. The HA is modified by being combined with active groups (such as amino groups and carboxyl groups of phospholipids) on the surface of the liposomes, and a low concentration of the HA will lead to insufficient modification coverage, a large number of liposomes without HA, and an inability to effectively realize active targeting and CD44 receptor combination. Meanwhile, astaxanthin is a fat-soluble and easily oxidized active ingredient, and the core function of the liposomes is to protect the stability of the astaxanthin. If the concentration of the HA is too high, the HA will compete for limited space to enter the double-layer membrane or the surface of the liposomes, so that the space for encapsulating the astaxanthin is occupied, the encapsulation rate of the liposomes is reduced, and the astaxanthin is leaked.

[0028] In another aspect, the application provides the hyaluronic acid modified astaxanthin liposomes in the application of the hyaluronic acid modified astaxanthin liposomes in any one of the above technical solutions in the preparation of an oral drug for treating CD44 overexpression diseases, and the molecular weight of the hyaluronic acid is 10-100 KD.

[0029] The technical solution limits the molecular weight of hyaluronic acid to 10-100KD to simultaneously consider the encapsulation efficiency of astaxanthin and the uptake efficiency of liver cancer cells; specifically, on the one hand, the spatial steric hindrance of HA with a molecular weight of 10-100KDa is small, the particle size of the modified liposome increases limitedly (usually <50nm), the interference with the lipid bilayer structure is small, and it is more easy to be stably combined with the surface of the liposome (through hydrophobic anchoring or chemical coupling), and is not easy to cause aggregation; on the other hand, HA with a molecular weight of 10-100KDa has strong permeability and can penetrate deep into tumor tissues.

[0030] It can be understood that the molecular weight of hyaluronic acid can also be 20KDa, 30KDa, 40KDa, 50KDa, 60KDa, 70KDa, 80KDa, 90Kda and any point value in the above range.

[0031] In some embodiments, the mass ratio of lecithin and phytosterol ester is 5-10:1.

[0032] Liposomes are nanoscale vesicles with low immunogenicity, non-toxicity and biodegradability, which can improve the loading capacity of different compounds and allow chemical modification. The chemical composition (phospholipid) and lipid bilayer structure of liposomes are very close to biological membranes, and have high biocompatibility. Among them, phytosterol ester has significant antioxidant activity, and phytosterol ester also has physiological functions such as neuroprotection, cholesterol reduction and liver protection. By using phytosterol ester to replace cholesterol and adding it to the lipid membrane to prepare liposome nanocarriers, not only can the negative effects of cholesterol intake on human health be avoided, but also phytosterol ester can form liposomes with lecithin to improve the stability and encapsulation efficiency of nanocarriers.

[0033] The above technical solution specifically limits the mass ratio of lecithin and phytosterol ester, which is beneficial to ensuring the structural integrity, biocompatibility and loading capacity of the liposome by sufficient lecithin, and enhancing the membrane stability and improving the retention and absorption efficiency of the functional components by appropriate phytosterol ester. In the liposome, on the one hand, lecithin is the main raw material of the liposome, and its molecule has a hydrophilic head and a hydrophobic tail, which needs to be self-assembled by a sufficient number of molecules to form a closed vesicle (liposome). The mass ratio of lecithin and phytosterol ester is set to 5-10:1, which can ensure the formation of a complete and continuous bilayer, avoid membrane rupture and vesicle collapse due to insufficient lecithin, or the inability to wrap astaxanthin; on the other hand, the molecular structure of phytosterol ester is similar to that of cholesterol, both of which are steroidal skeletons that can be embedded in the lecithin bilayer to provide support. Appropriate amount of phytosterol ester can reduce the fluidity between lecithin molecules, enhance the tolerance of the membrane to the external environment, and avoid leakage of the contents due to insufficient membrane stability of the liposome. However, if the proportion of phytosterol ester is too high, it will squeeze the lecithin molecules too much, destroy the ordered arrangement of the bilayer, and even lead to membrane structure disorder and vesicle rupture. If the proportion is too low, it cannot play its supporting and stabilizing role, and the liposome is prone to instability due to excessive membrane fluidity.

[0034] It can be understood that the mass ratio of lecithin and phytosterol ester can also be 6:1, 7:1, 8:1, 9:1, and any point value ratio within the above range.

[0035] It should be noted that the size of the hyaluronic acid molecular weight and the composition ratio of the liposome membrane material have certain synergies and mutual cooperation to ensure the embedding rate and modification effect of the liposome. Specifically, the mass ratio of the size of the hyaluronic acid molecular weight and the mass ratio of the egg phospholipid and the phytosterol ester should be matched to avoid the modification of the hyaluronic acid from destroying the integrity of the liposome bilayer, and to ensure that the hyaluronic acid can be stably anchored on the membrane surface. Among them, the molecular weight of hyaluronic acid determines its chain length and spatial conformation: low molecular weight hyaluronic acid has short chain and compact conformation; high molecular weight hyaluronic acid has long chain and loose conformation, and is easy to form a winding spatial structure. When the proportion of phytosterol ester in the liposome is low, the content of phytosterol ester is low, and after inserting into the hydrophobic region of the phospholipid bilayer, the inhibition of membrane fluidity is weak, the lateral movement of phospholipid molecules is more active, and the hydrophilic head (phosphate group) on the membrane surface is relatively loose. At this time, the low molecular weight hyaluronic acid should be matched for modification, and the hydrophilic group carried by the low molecular weight hyaluronic acid can form effective combination (through weak hydrogen bond) with the loose arrangement of the phospholipid head on the membrane surface, and the short chain will not produce excessive pulling on the liposome membrane. When the proportion of phytosterol ester in the liposome is high, the content of phytosterol ester is increased, and a large amount of phytosterol ester is inserted into the phospholipid bilayer to play a role similar to a scaffold, limit the movement of phospholipid molecules, and make the membrane as a whole more rigid. At the same time, due to the space extrusion of phytosterol ester, the arrangement of the phospholipid head is more compact, and the anti-deformation ability of the membrane is enhanced. At this time, the high molecular weight hyaluronic acid can be matched for modification, and the modification effect will not be affected due to the pulling force of the high molecular weight hyaluronic acid on the surface of the liposome.

[0036] In some embodiments, the mass ratio of egg phospholipid and phytosterol ester is 5-10:1, the molecular weight of hyaluronic acid is 10-100KD, and as the mass ratio of phytosterol ester increases, the molecular weight of the selected hyaluronic acid increases.

[0037] In some embodiments, the particle size of the hyaluronic acid modified astaxanthin liposome is 130-160nm, PDI<0.4, and Zeta≤-27.8mV, indicating that the dispersion system has good stability.

[0038] The application also provides a preparation method of the hyaluronic acid modified astaxanthin liposome described in any of the above technical solutions, which comprises: mixing and dissolving hyaluronic acid, N-hydroxysuccinimide and 1-ethyl-3-(3 dimethylaminopropyl) carbodiimide to obtain a uniform solution, mixing the uniform solution with DSPE-PEG 2000 -NH2 dissolved in dichloromethane, stirring at room temperature, filtering to obtain a viscous solution, evaporating the solvent, and further precipitating by adding acetone, and drying the precipitate to obtain DSPE-PEG 2000 -HA.

[0039] Specifically, HA, N-hydroxysuccinimide and 1-ethyl-3-(3 dimethylaminopropyl) carbodiimide are dissolved in distilled water, stirred at 4℃ for 4h, the obtained uniform solution is mixed with DSPE-PEG 2000 -NH2 dissolved in dichloromethane (DCM), stirred at room temperature for 12h, a thick solution is obtained by filtration, the solvent is evaporated, and further precipitation is performed by adding acetone, the precipitate is filtered and dried in vacuum to obtain DSPE-PEG 2000 -HA.

[0040] In some embodiments, further comprising: adding lecithin and phytosterol ester into chloroform, then adding astaxanthin-chloroform stock solution, obtaining a liposome film after evaporation under reduced pressure, adding ultrapure water to completely dissolve the film, and obtaining Lip-AST by crushing and extruding; mixing Lip-AST with DSPE-PEG 2000 -HA solution (DPSPE-PEG 2000 -HA powder dissolved in ultrapure water) to obtain a uniform solution, and obtaining hyaluronic acid modified astaxanthin liposomes by water bath reaction.

[0041] The above technical solution adopts a film hydration-ultrasonic method to prepare astaxanthin liposomes (Lip-AST). Specifically, 100mg of egg yolk lecithin and 20mg of phytosterol ester are added into chloroform for dissolution, then 2ml of astaxanthin-chloroform stock solution with a concentration of 1mg / mL is added, a liposome film is obtained after evaporation under reduced pressure at 40℃, 10mL of ultrapure water is added for vigorous shaking until the film is completely dissolved, and the film is treated by ultrasonic cell disruption, and then extruded through a membrane with a diameter of 0.45μm to obtain Lip-AST.

[0042] The above technical solution adopts a post-insertion method for HA modification. Specifically, 1mL of Lip-AST is mixed with DSPE-PEG 2000 -HA solution with different film-forming material proportions (i.e. 0.6mg / mL, 1.2mg / mL, 1.8mg / mL, 2.4mg / mL), and the mixture is uniformly mixed and incubated in a water bath at 50℃ for 2h, and then filtered through a membrane with a diameter of 0.22μm to obtain astaxanthin liposomes with different HA modification proportions, i.e. 5%HA@Lip, 10%HA@Lip, 15%HA@Lip, and 20%HA@Lip.

[0043] The encapsulation rate of astaxanthin in the hyaluronic acid modified astaxanthin liposomes prepared by the above preparation method is ≥77%, wherein the encapsulation rate of astaxanthin (%) = the mass of astaxanthin in the carrier / the total amount of astaxanthin input × 100.

[0044] The application further provides an oral drug for treating CD44 overexpression diseases, comprising the hyaluronic acid modified astaxanthin liposome in any of the above technical solutions.

[0045] In order to more clearly and specifically introduce the hyaluronic acid modified astaxanthin liposome, the preparation method, application and oral drug for treating CD44 overexpression diseases provided by the embodiments of the application, the following will be described in combination with specific embodiments.

[0046] Embodiment 1 1, DSPE-PEG 2000 -HA synthesis As Figure 1 , DSPE-PEG 2000 -HA is synthesized by amine coupling of hyaluronic acid (HA) and DSPE-PEG 2000 -NH2. 10KDa HA, N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide are dissolved in distilled water, and stirred at 4℃ for 4h. The obtained uniform solution is mixed with DSPE-PEG 2000 -NH2 dissolved in dichloromethane (DCM), and stirred at room temperature for 12h. The obtained viscous solution is filtered, and the solvent is evaporated, and further precipitated by adding acetone. The precipitate is filtered and vacuum dried to obtain DSPE-PEG 2000 -HA.

[0047] 2, Preparation of HA modified astaxanthin liposome The astaxanthin liposome (Lip-AST) is prepared by the method of film hydration-ultrasonic. 100mg of egg yolk lecithin and 20mg of phytosterol ester are dissolved in chloroform, and then 2mL of astaxanthin-chloroform stock solution with a concentration of 1mg / mL is added. The liposome film is obtained after evaporation at 40℃ under reduced pressure. 10mL of ultrapure water is added and shaken vigorously until the film is completely detached and dissolved. The ultrasonic cell disrupter is used for ultrasonic treatment, and then extruded through a membrane with a diameter of 0.45μm to prepare Lip-AST. The post-insertion method is used for HA modification. 1mL of Lip-AST is mixed with 0.6mg / mL of DSPE-PEG 2000 -HA solution, and constant temperature water bath at 50℃ for 2h, and then filtered through a membrane with a diameter of 0.22μm to obtain astaxanthin liposome with a HA modification ratio of 5%, i.e. 5%HA@Lip.

[0048] Embodiment 2 The same as Embodiment 1, except that 1mL of Lip-AST is mixed with 1.2mg / mL of DSPE-PEG 2000The HA solution was mixed uniformly and placed in a constant temperature water bath at 50°C for 2h, and then filtered through a 0.22μm membrane to obtain astaxanthin liposomes with a 10% HA modification ratio, i.e., 10%HA@Lip.

[0049] Example 3 The same as in Example 1, except that 1 mL of Lip-AST was mixed with 1.8 mg / mL of DSPE-PEG 2000 The HA solution was mixed uniformly and placed in a constant temperature water bath at 50°C for 2h, and then filtered through a 0.22μm membrane to obtain astaxanthin liposomes with a 15% HA modification ratio, i.e., 15%HA@Lip.

[0050] Example 4 The same as in Example 1, except that 1 mL of Lip-AST was mixed with 2.4 mg / mL of DSPE-PEG 2000 The HA solution was mixed uniformly and placed in a constant temperature water bath at 50°C for 2h, and then filtered through a 0.22μm membrane to obtain astaxanthin liposomes with a 20% HA modification ratio, i.e., 20%HA@Lip.

[0051] Comparative Example 1 A 1mg / mL astaxanthin-chloroform stock solution was prepared using DMSO, and then diluted with a phosphate buffer (PBS) to 0.2mg / mL to obtain a free astaxanthin (AST) solution.

[0052] Comparative Example 2 Astaxanthin liposomes were prepared by a thin film hydration-ultrasonic method. 100mg of egg yolk lecithin and 20mg of phytosterol ester were dissolved in chloroform, and then 2mL of a 1mg / mL astaxanthin-chloroform stock solution was added. After evaporation under reduced pressure at 40°C, a thin film of the liposomes was obtained. 10mL of ultrapure water was added and shaken vigorously until the film was completely dissolved. The resulting solution was treated with an ultrasonic cell disruptor, and then filtered through a 0.45μm membrane to obtain astaxanthin liposomes without any modification (Lip-AST).

[0053] Performance Test 1. DSPE-PEG 2000 HA Characterization HA and DSPE-PEG 2000 and DSPE-PEG 2000 HA was characterized by Fourier transform infrared spectroscopy (FT-IR) to characterize its structure, and the results are shown in Figure 2 .

[0054] The results show that the HA has a characteristic absorption peak at 1640cm -1with characteristic peaks at 1740 cm -1 (C-O-C), 1077 cm - 1(C-O) also have characteristic absorption peaks. DSPE-PEG 2000 at 2888 cm -1 and 2917 cm -1 are characteristic absorption peaks of CH alkyl; 1741 cm -1 is the absorption peak of carbonyl copper. DSPE-PEG 2000 -HA Fourier transform infrared spectrogram, 1740 cm -1 , 2884 cm -1 , 2920 cm -1 correspond to the characteristic absorption peaks of DSPE-PEG 2000 ; 1049 cm -1 , 1081 cm -1 , 1647 cm -1 correspond to the characteristic absorption peaks of HA. The above results show that DSPE-PEG 2000 -HA is successfully synthesized.

[0055] 2. Physicochemical property characterization of HA modified astaxanthin liposomes The particle size of the HA modified astaxanthin liposomes prepared in Examples 1-4 was determined, and the results are shown in Table 1; the Zeta potential of the HA modified astaxanthin liposomes prepared in Examples 1-4 was determined, and the results are shown in Table 1; the surface morphology of the liposomes was observed by transmission electron microscopy (TEM), and the results are shown in Table 1. Figures 3-6 Figures 7-10 The above results show that the particle size of the astaxanthin liposomes modified by HA at different concentrations ranges from 130-160 nm, the PDI is <0.4, and the Zeta potential is about -30 mV; the results show that the dispersion system has good stability.

[0056] Table 1 Zeta potential results of HA modified astaxanthin liposomes

[0057]

[0058] 3. Determination of HA content in hyaluronic acid modified astaxanthin liposomes ​​First draw the standard curve of HA. Precision weighing HA standard, preparation of 400 μg / mL HA solution, respectively 0.0, 0.25, 0.5, 0.6, 0.7, 0.8, 0.9 mL standard control solution, all add water to 1.0 mL mixed, then cooled to 4℃ below with ice water. Under the shaking, drop 5.0 mL of 0.025 mol / L borax sulfuric acid solution, mix well, then heated in boiling water bath for 15 min, cooled to room temperature. Then add 0.5 mL of carbazole test solution, shake well, heat in boiling water bath for 15 min, cool to room temperature, then measure the absorbance. The content of HA on the liposome was determined by carbazole method, and 1 mL of hyaluronic acid modified astaxanthin liposome suspension in examples 1-4 was taken, cooled to 4℃ in ice water bath, and the subsequent steps were the same as the determination of the above standard.

[0059] As shown in Figure 11 , the content of HA in astaxanthin liposomes with HA modification ratio of 5%, 10%, 15% and 20% was 320.48±34.71 μg / mL, 374.20±30.47 μg / mL, 417.20±14.65 μg / mL and 481.20±25.71 μg / mL respectively. And with the increase of HA concentration, the content of HA on the surface of liposome was higher.

[0060] 4. Determination of encapsulation efficiency From examples 1-4, 1 mL of liposome suspension was mixed with dichloromethane-methanol mixed solution (volume ratio of 4:2) by vortex oscillation, then centrifuged at 10℃ (8000 rpm, 10 min), and the lower organic phase component was collected. Dichloromethane was used as a blank solution, and the absorbance value was determined at 480 nm by spectrophotometry to determine the AST content.

[0061] Encapsulation efficiency (%) = mass of astaxanthin in carrier / total amount of astaxanthin input × 100.

[0062] As shown in Figure 12 , the insertion of HA had an effect on the effective encapsulation of AST, and the encapsulation efficiency of astaxanthin liposomes with HA modification ratio of 5%, 10%, 15% and 20% was 84.80±1.51%, 82.80±1.26%, 79.50±1.30% and 77.60±1.43% respectively.

[0063] The results showed that: with the increase of HA concentration, the encapsulation efficiency of liposome showed a decreasing trend. This may be due to the large size of HA molecules and the steric hindrance effect, with the increase of concentration, more HA molecules will compete for the limited space into the bilayer membrane or surface of liposome, resulting in the space for encapsulating astaxanthin being occupied, which reduces the encapsulation efficiency.

[0064] 5. Storage stability The particle size, PDI and Zeta potential of the HA modified astaxanthin liposomes were measured after 30 days of storage at 4°C to evaluate their storage stability. The results are shown in Table 2 and Figures 13-15 Fig. 1, where the particle size of 10% HA@Lip and 20% HA@Lip at 30 days was significantly different from that at the first day, and the PDI change also had the same trend; the particle size of 5% HA@Lip and 15% HA@Lip did not change significantly after 30 days of storage. The absolute value of the Zeta potential of each group decreased significantly (***p < 0.001), but was still greater than 20 mV. Studies have reported that the absolute value of the Zeta potential of a nano-carrier is greater than 25 mV when it has good stability.

[0065] The above results show that 15% HA@Lip has the best stability after 30 days of storage, with a decrease in particle size, no significant change in PDI, and an absolute value of the Zeta potential greater than 25 mV.

[0066] Table 2 Stability of HA modified astaxanthin liposomes after 30 days of storage

[0067] 6. In vitro antioxidant capacity The in vitro antioxidant activity of HA modified astaxanthin liposomes was evaluated by measuring the DPPH free radical scavenging activity. 0.5 mL of 5%, 10%, 15%, and 20% HA@Lip liposomes or free AST solution (0.2 mg / mL) was mixed with 1 mL of DPPH ethanol solution (0.1 mmol / L) by vortex oscillation for 30 s, and then left to stand in the dark for 30 min. The mixture was centrifuged at 5000 rpm at room temperature for 10 min, and the supernatant was collected. The absorbance (OD 517 ) was measured using a microplate reader. An equal volume of deionized water was used as a blank group, and an equal volume of anhydrous ethanol was used as a control group.

[0068] The DPPH free radical scavenging rate (%) was calculated as follows: DPPH free radical scavenging rate (%) = [1 - (OD test - OD control ) / OD blank ] x 100 As Figure 16The DPPH radical scavenging activity of the HA modified astaxanthin liposomes was significantly improved compared with free AST ( ** p < 0.01). Among them, the DPPH radical scavenging rate of liposomes with different HA modification ratios increased in turn. HA has a certain antioxidant effect, which may be due to the synergistic antioxidant effect of HA and astaxanthin. The DPPH radical scavenging rates of free AST, 5%HA@Lip, 10%HA@Lip, 15%HA@Lip and 20%HA@Lip were 9.98 ± 3.67%, 52.20 ± 23.41%, 56.34 ± 5.36%, 58.95 ± 4.95% and 60.48 ± 1.63%, respectively.

[0069] 7. In vitro simulated digestion The simulated digestion solution was prepared according to the method of Brodkorb, and the whole experiment was carried out at 37°C.

[0070] Preparation method of digestion solution: Simulated saliva: potassium chloride (37.3 g / L), potassium dihydrogen phosphate (68 g / L), sodium bicarbonate (42 g / L), magnesium chloride hexahydrate (30.5 g / L), ammonium carbonate (48 g / L), hydrochloric acid (6 mol / L), anhydrous calcium chloride (44.1 g / L), and 75000 U / L salivary amylase dissolved in deionized water, mixed uniformly and adjusted to pH 7.0.

[0071] Simulated gastric juice: potassium chloride (37.3 g / L), potassium dihydrogen phosphate (68 g / L), sodium bicarbonate (42 g / L), sodium chloride (117 g / L), magnesium chloride hexahydrate (30.5 g / L), ammonium carbonate (48 g / L), hydrochloric acid (6 mol / L), anhydrous calcium chloride (44.1 g / L), and 2000000 U / L pepsin dissolved in deionized water, mixed uniformly and adjusted to pH 3.0.

[0072] Simulated intestinal juice: potassium chloride (37.3 g / L), potassium dihydrogen phosphate (68 g / L), sodium bicarbonate (42 g / L), sodium chloride (117 g / L), magnesium chloride hexahydrate (30.5 g / L), hydrochloric acid (6 mol / L), anhydrous calcium chloride (44.1 g / L), and 100000 U / L trypsin, 10 mM cholic acid dissolved in deionized water, mixed uniformly and adjusted to pH 7.0.

[0073] Take 5 mL saliva and liposome sample (15% HA@Lip prepared in Example 3) or free AST solution (Comparative Example 1) (v:v = 1:1) to simulate oral digestion for 5 min; add gastric juice 10 mL successively, digest for 2 h, then add intestinal juice 20 mL, digest for 2 h, and carry out gastrointestinal digestion simulation. Centrifuge at 8000 rpm for 10 min after sampling at intervals, take the supernatant to obtain the AST micelle layer, and calculate the conversion value and bioavailability according to the formula: Astaxanthin conversion value (%) = m / M x 100% In the formula: m is the astaxanthin content after digestion (mg), and M is the total amount of initial astaxanthin added (mg).

[0074] The results are shown in Table 1. Figure 17 As shown in Table 1, the conversion value of HA modified astaxanthin liposome at the end of digestion is 56.86%, which is 1.83 times that of free AST. This is because the liposome has good water solubility, while the solubility of free AST is low, and it is easy to aggregate and settle during oral, gastric and intestinal digestion, and adhere to the inner wall of the oral cavity and gastrointestinal tract, resulting in a lower conversion value. In addition, lecithin and phytosterol as the protective layer of the liposome effectively reduces the degradation of AST in the digestive juice, improves the bioavailability, and increases the conversion value.

[0075] 8. In vitro cytotoxicity The in vitro cytotoxicity of HA modified astaxanthin liposome was evaluated by determining the survival rate of hek-293 cells by CCK8 method. Cells were seeded in a 96-well plate at a density of 5 x 10 4 cells / well and incubated overnight, and then 5% HA@Lip (Example 1), 10% HA@Lip (Example 2), 15% HA@Lip (Example 3), 20% HA@Lip (Example 4) or free AST (Comparative Example 1) were added respectively, and incubated for 24 h. Then 10 μL of CCK-8 solution was added to each well and incubated for 2 h. The blank well was culture medium without cells, and the control well was cells without drugs. The absorbance at 450 nm was measured by a microplate reader.

[0076] Cell survival rate = (As-Ab) / (Ac-Ab) x 100% Wherein, As: experimental well, Ab: blank well, Ac: control well.

[0077] The results are shown in Table 1. Figure 18 As shown in Table 1, the conversion value of HA modified astaxanthin liposome at the end of digestion is 56.86%, which is 1.83 times that of free AST. This is because the liposome has good water solubility, while the solubility of free AST is low, and it is easy to aggregate and settle during oral, gastric and intestinal digestion, and adhere to the inner wall of the oral cavity and gastrointestinal tract, resulting in a lower conversion value. In addition, lecithin and phytosterol as the protective layer of the liposome effectively reduces the degradation of AST in the digestive juice, improves the bioavailability, and increases the conversion value.

[0078] 9. In vitro uptake of hepatocellular carcinoma cells The uptake of astaxanthin liposomes with different HA modification ratios by liver cancer cells (huh-7) overexpressing CD44 receptors was investigated. To facilitate visual observation, coumarin 6 (C6) was used instead of AST to prepare liposome samples, resulting in C6@Lip, 5%HA@Lip-C6, 10%HA@Lip-C6, 15%HA@Lip-C6, and 20%HA@Lip-C6. Huh-7 cells were seeded in a six-well plate at a density of 5×10 5 Each group of liposomes was added and incubated at 37°C for 2 h. The cells were fixed with 4% paraformaldehyde and washed with PBST three times. DAPI was diluted in a PBS solution containing 1% BSA and 2% triton X-100, and 500 μL was added to each well. The cells were incubated in the dark for 1-2 h, washed with PBST three times, and observed under a fluorescence microscope. A competitive inhibition group was also set up, in which the cells were pre-incubated with 0.2 mg / mL HA for 2 h before the addition of the liposome sample (20%HA@Lip-C6). The fluorescence intensity was counted, Figure 19 The cell uptake rate of each group was calculated based on the unmodified astaxanthin liposomes.

[0079] In combination Figure 19 , 20 It was found that green fluorescence signals were detected in huh-7 cells, and the cell uptake rate calculated based on the fluorescence signal intensity was in the order of 15%HA@Lip-C6, 10%HA@Lip-C6, 5%HA@Lip-C6, 20%HA@Lip-C6, 20%HA@Lip-C6 (HA pretreated), C6@Lip, and C6 from high to low. The cell uptake rate of the four HA modification ratio liposomes was higher than that of the unmodified HA liposomes, indicating that HA can specifically recognize the overexpressed CD44 receptors on liver cancer cells, thereby increasing the cell uptake rate. Moreover, the cell uptake rate of the HA modified liposomes was significantly higher than that of the free C6 group (**p<0.01, ***p<0.001). To further verify the targeting effect of HA, an HA pretreatment group was set up, and the results showed that after the CD44 receptor was blocked by HA, the cell uptake rate of 20%HA@Lip was significantly reduced (#p<0.05).

[0080] This result further indicates that HA modified astaxanthin liposomes can effectively deliver astaxanthin to liver cancer cells by targeting CD44 receptors, significantly increasing the intracellular astaxanthin content.

[0081] 10. In vitro inhibition of liver cancer cell activity The survival rate of huh-7 cells was determined by CCK8 to evaluate the in vitro liver cancer cell inhibition of HA modified astaxanthin liposomes. The cells were seeded in a six-well plate at a density of 5×104 The density of 1 cell / hole was seeded in a 96-well plate overnight, and 5% HA@Lip (Example 1), 10% HA@Lip (Example 2), 15% HA@Lip (Example 3), 20% HA@Lip (Example 4) or free AST (Comparative Example 1) was added respectively, and incubated for 24 h. Then 10 μL CCK-8 solution was added to each well for a total of 2 h of incubation. The blank well was culture medium without cells, and the control well was cells without drugs. The absorbance at 450 nm was determined by a microplate reader.

[0082] Cell survival rate = (As-Ab) / (Ac-Ab) x 100% Wherein, As: experimental hole, Ab: blank hole, Ac: control hole.

[0083] The results are shown in Figure 21 AST, 5% HA@Lip, 10% HA@Lip, 15% HA@Lip, 20% HA@Lip on huh-7 liver cancer cell inhibition rate in turn 13.80 ± 1.04, 20.82 ± 1.47, 23.28 ± 0.97, 26.81 ± 1.19, 22.16 ± 0.69.

[0084] In summary, the present application uses hyaluronic acid and phospholipid material coupling, with lecithin and phytosterol ester as wall material, to prepare a new type of HA modified liposome for delivering astaxanthin. The results show that the particle size of the liposome is in the range of 130-160 nm, the PDI is 0.1-0.4, the Zeta potential is about -30 mV, and it has good encapsulation efficiency and storage stability. With the increase of the content of HA on the surface of the liposome, the encapsulation efficiency shows a downward trend, and the antioxidant performance shows an upward trend. During the in vitro simulated digestion, the conversion value of the HA modified astaxanthin liposome is significantly higher than that of free AST, effectively improving its oral bioavailability. In vitro cell experiments show that astaxanthin liposome has no obvious cytotoxicity and good safety. Compared with free AST, the cell uptake rate of HA modified astaxanthin liposome is significantly increased, indicating that the drug delivery carrier has targeting property to liver cancer cells. The construction of the new astaxanthin targeted delivery system and the in vitro action research of astaxanthin effectively improve the bioavailability of astaxanthin and the targeting property to liver cancer cells.

Claims

1. The application of a hyaluronic acid-modified astaxanthin liposome in the preparation of an oral drug for treating CD44 overexpression diseases, characterized in that, Hyaluronic acid-modified astaxanthin liposomes deliver astaxanthin to CD44 receptor-overexpressing liver cancer cells via hyaluronic acid binding to the CD44 receptor. Hyaluronic acid-modified astaxanthin liposomes are delivered orally using hyaluronic acid-modified liposomes as carriers of astaxanthin. Liposomes include lecithin and phytosterol esters; and hyaluronic acid-modified astaxanthin liposomes use lecithin and phytosterol esters as wall materials.

2. The application of the hyaluronic acid-modified astaxanthin liposomes according to claim 1 in the preparation of oral drugs for treating CD44 overexpression diseases, characterized in that, Hyaluronic acid and DSPE-PEG 2000 Amine coupling to synthesize DSPE-PEG 2000 -HA; Astaxanthin is encapsulated by lecithin and phytosterol esters, and then further processed by DSPE-PEG. 2000 -HA modification yielded hyaluronic acid-modified astaxanthin liposomes.

3. The application of the hyaluronic acid-modified astaxanthin liposomes according to claim 2 in the preparation of oral drugs for treating CD44 overexpression diseases, characterized in that, DSPE-PEG in hyaluronic acid-modified astaxanthin liposomes 2000 The content of -HA is 285-507 μg / mL.

4. The application of the hyaluronic acid-modified astaxanthin liposomes according to claim 3 in the preparation of oral drugs for treating CD44 overexpression diseases, characterized in that, DSPE-PEG in hyaluronic acid-modified astaxanthin liposomes 2000 The content of -HA was 417.20±14.651μg / mL.

5. The hyaluronic acid-modified astaxanthin liposomes according to any one of claims 1-4 in the preparation of an oral medicament for treating CD44 overexpression diseases, characterized in that, Hyaluronic acid has a molecular weight of 10-100 kDa.

6. The hyaluronic acid-modified astaxanthin liposomes according to claim 5, characterized in that, The mass ratio of lecithin to phytosterol esters is 5-10:

1.

7. The hyaluronic acid-modified astaxanthin liposomes according to claim 5, characterized in that, The particle size is 130-160 nm, PDI < 0.4, and Zeta ≤ -27.8 mV.

8. The method for preparing hyaluronic acid-modified astaxanthin liposomes according to any one of claims 5-7, characterized in that, include: Hyaluronic acid, N-hydroxysuccinimide, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide were mixed and dissolved to obtain a homogeneous solution. The homogeneous solution was then mixed with DSPE-PEG dissolved in dichloromethane. 2000 -NH2 was mixed and stirred at room temperature. The mixture was filtered to obtain a viscous solution. The solvent was evaporated, and acetone was added for further precipitation. The precipitate was dried to obtain DSPE-PEG. 2000 -HA.

9. The method for preparing hyaluronic acid-modified astaxanthin liposomes according to claim 8, characterized in that, Also includes: Lecithin and phytosterol esters were dissolved in chloroform, then astaxanthin-chloroform stock solution was added, and the mixture was evaporated under reduced pressure to obtain a liposome film. Ultrapure water was added until the film was completely detached and dissolved. After crushing, the film was extruded to prepare Lip-AST. Lip-AST with DSPE-PEG 2000 - The HA solution was mixed thoroughly and reacted in a water bath to obtain hyaluronic acid-modified astaxanthin liposomes.

10. An oral medication for treating CD44 overexpression diseases, characterized in that, Astaxanthin liposomes modified with hyaluronic acid as described in any one of claims 5-7.

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