Drug-loaded liposome based on colon targeting and preparation method thereof

Abstract

The invention discloses a drug-loaded liposome based on colon targeting and a preparation method thereof, and belongs to the technical field of nano targeting drugs, the drug-loaded liposome sequentially comprises an active ingredient inner core, a chitosan interlayer and a sodium alginate shell layer from inside to outside; the preparation method comprises the following steps: preparing an active ingredient core solution; under a stirring condition, dropwise adding the active ingredient core solution into the chitosan solution, and continuously stirring after dropwise adding to obtain an active ingredient-chitosan solution; the preparation method comprises the following steps: dropwise adding an effective component chitosan solution into a sodium alginate solution under a stirring condition, and continuously stirring after dropwise adding is finished, so as to obtain the chitosan-sodium alginate hydrogel. The lipidosome is stable in structure in the stomach and the small intestine and high in gastric acid damage resistance, the targeting property of the medicine in the colon can be effectively improved, the utilization rate of the medicine is increased, and the problem that the content of effective components, acting on the colon, of an existing medicine is low is solved.

Description

Technical Field

[0001] The invention belongs to the technical field of nano-targeted drugs, and in particular relates to a colon-targeted drug-loaded liposome and a preparation method and application thereof. Background Art

[0002] Ulcerative colitis (UC) is characterized by spontaneous inflammation of the gastrointestinal tract, characterized by recurrence and remission of mucosal inflammation, and patients often have bloody stools and diarrhea. With the improvement of living conditions and changes in residents' dietary intake, the incidence of ulcerative colitis has gradually increased. A large number of studies have shown that the pathogenic mechanism of ulcerative colitis is determined by many factors, including: individual genetic susceptibility, external environment, intestinal microbial flora, and immune response are all related to its pathogenesis.

[0003] The abundance of Clostridium and Bacteroides is prone to decrease in the intestines of patients with ulcerative colitis. The proportion of Firmicutes and Clostridium that produce short-chain fatty acids in the intestines decreases. At the same time, the number of probiotics decreases significantly, while the number of pathogenic bacteria increases. The enterotoxins released by them can destroy the integrity of the intestinal epithelium and increase the permeability of epithelial cells. At the same time, due to the reduced diversity of intestinal flora, the number of bacteria that release inflammatory mediators in the intestine increases, thereby activating the immune activity of the intestinal mucosa and producing a persistent local inflammatory response. To date, there is no effective treatment for ulcerative colitis, and the recurrence rate is high. Commonly used clinical drugs include anti-inflammatory drugs (such as 5-aminosalicylic acid, corticosteroids) and immunosuppressants (such as azathioprine and 6-mercaptopurine).

[0004] Due to the particularity of the location of onset of ulcerative colitis, therapeutic drugs are required to be released in the colon to improve the effectiveness of the drugs. Oral drugs must pass through the stomach, and most of the active ingredients are destroyed by gastric acid in the stomach, thereby weakening the therapeutic effect of the drugs. Therefore, it is particularly important to reduce the amount of drugs destroyed in the stomach. Summary of the invention

[0005] In view of the above-mentioned deficiencies in the prior art, the present invention provides a colon-targeted drug-loaded liposome, which has a stable structure in the stomach and small intestine and a strong resistance to gastric acid damage. It can effectively improve the targeting of drugs in the colon, improve drug utilization, and solve the problem of low drug content acting on the colon in the prior art.

[0006] To achieve the above purpose, the technical solution adopted by the present invention to solve the technical problem is:

[0007] A colon-targeted drug-loaded liposome, which comprises an active ingredient inner core, a chitosan interlayer and a sodium alginate shell layer from the inside out.

[0008] Furthermore, the active ingredient is a mixture of honokiol and rhein, and the mass ratio of honokiol to rhein is 0.3-1.8:1.

[0009] The preparation method of the colon-targeted drug-loaded liposomes comprises the following steps:

[0010] (1) preparing an active ingredient core solution;

[0011] (2) under stirring conditions, the active ingredient core solution is added dropwise to the chitosan solution, and stirring is continued for 0.5-2 hours after the addition is completed to obtain an active ingredient-chitosan solution;

[0012] (3) Under stirring conditions, the active ingredient-chitosan solution is added dropwise to the sodium alginate solution, and stirring is continued for 0.5-2 hours after the addition is completed to obtain the obtained product.

[0013] In the above-mentioned method for preparing colon-targeted drug-loaded liposomes, the active ingredient core solution in step (1) is prepared by the following method:

[0014] (1) adding a mixture of active ingredients, soybean lecithin and cholesterol into an organic solvent, dissolving under heating and ultrasonic conditions to obtain a mixed solution, and then evaporating and removing the organic solvent to obtain a drug-loaded phospholipid film;

[0015] (2) PBS buffer is added to the drug-loaded phospholipid film to hydrate the drug-loaded phospholipid film, ultrasonically disrupted, and then filtered to obtain the drug-loaded phospholipid film.

[0016] Furthermore, in step (1), the mass ratio of the active ingredient, soybean lecithin and cholesterol is 5-8:50-80:10-16.

[0017] Furthermore, in step (2), the volume mass ratio of PBS buffer to phospholipid is 2-5:1.

[0018] Furthermore, the hydration time in step (2) is 10-40 min.

[0019] Furthermore, in step (2), the ultrasonic power is 500-700 W, and the ultrasonic time is 3-12 min.

[0020] Furthermore, in step (2), the concentration of the chitosan solution is 0.4-4 mg / mL, and the volume of the active ingredient core solution is the same as that of the chitosan solution.

[0021] Furthermore, in step (3), the concentration of the sodium alginate solution is 1.3-3 mg / mL, and the volume of the active ingredient-chitosan solution is the same as that of the sodium alginate solution.

[0022] The beneficial effects produced by the present invention are:

[0023] 1. In the present invention, honokiol and rhein are prepared into the form of liposomes, which can improve the solubility of both, so that the encapsulation rate of the drug can reach more than 80%, thereby improving the bioavailability of the drug.

[0024] 2. The drug-loaded liposomes in the present invention are colon-targeted, and oral administration can prevent the drug from being degraded in the upper digestive tract, thereby improving the bioavailability of the drug; specifically, in the present invention, the liposomes are combined with the positively charged chitosan through layer-by-layer electrostatic adsorption, and then wrapped with a layer of negatively charged sodium alginate. Sodium alginate is difficult to decompose in the stomach and small intestine, but can be decomposed by microorganisms in the colon, which can protect the flora and target the drug. Therefore, the liposomes coated with chitosan and sodium alginate can reduce degradation in the stomach and small intestine environment, enhance the stability of the liposome system, and can also prolong the drug release time and improve the bioavailability. In addition, the polysaccharide raw materials chitosan and sodium alginate have the advantages of wide sources, high biosafety, low cost, and easy functional modification, and have unique application advantages and potential in the development of targeted delivery systems for colon diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is a particle size distribution diagram of the liposomes in this application;

[0026] Figure 2 This is the appearance diagram of the lipid dispersion in this application;

[0027] Figure 3 is the in vitro release curve of magnolol (HNK) in liposomes when the medium pH is 1.2;

[0028] Figure 4 is the in vitro release curve of magnolol (HNK) in liposomes when the medium pH is 6.8;

[0029] Figure 5 is the in vitro release curve of magnolol (HNK) in liposomes when the medium pH is 7.8;

[0030] Figure 6 The in vitro release curve of rhein (RH) in liposomes when the medium pH is 1.2

[0031] Figure 7 The in vitro release curve of rhein (RH) in liposomes when the medium pH is 6.8

[0032] Figure 8 The in vitro release curve of rhein (RH) in liposomes when the medium pH is 7.8

[0033] Fig. 9 The colon appearance of mice in different groups;

[0034] Fig.10 It is the MPO statistical graph of mice after treatment;

[0035] Fig.11 Statistical graph of IL6 in mice after treatment. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical scheme and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention, that is, the embodiments described are only part of the embodiments of the present invention, rather than all of the embodiments.

[0037] Therefore, the following detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of the present invention.

[0038] It should be noted that relational terms such as "first" and "second" are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprises" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

[0039] The features and performance of the present invention are further described in detail below in conjunction with the embodiments and drawings.

[0040] Example 1

[0041] A colon-targeted drug-loaded liposome, the drug-loaded liposome comprising an active ingredient core, a chitosan interlayer and a sodium alginate shell layer from the inside out, wherein the active ingredient is a mixture of honokiol and rhein, and the mass ratio of honokiol to rhein is 0.5:1;

[0042] The preparation method of the colon-targeted drug-loaded liposomes comprises the following steps:

[0043] (1) adding a mixture of active ingredient, soybean lecithin and cholesterol to an organic solvent, wherein the mass ratio of active ingredient, soybean lecithin and cholesterol is 5:50:10, dissolving under heating and ultrasonic conditions to obtain a mixed solution, and then removing the organic solvent by rotary evaporation at 50° C. and 130 rpm to obtain a drug-loaded phospholipid film; adding 150 mL of PBS buffer solution with a pH of 7.4 to the drug-loaded phospholipid film to hydrate the drug-loaded phospholipid film for 30 minutes, and then ultrasonically crushing the film at 600 W for 8 minutes, and then filtering with a 0.45 μm filter membrane to obtain an active ingredient core solution;

[0044] (2) under stirring conditions of 400 r / min, the active ingredient core solution was added dropwise to a chitosan solution having a concentration of 1.5 mg / mL at a volume ratio of 1:1, and stirring was continued for 1 h after the addition was completed to obtain an active ingredient-chitosan solution;

[0045] (3) Under stirring conditions of 400 r / min, the active ingredient-chitosan solution was added dropwise to a sodium alginate solution with a concentration of 1.5 mg / mL in a volume ratio of 1:1, and stirring was continued for 12 hours after the addition was completed.

[0046] Example 2

[0047] A colon-targeted drug-loaded liposome, the drug-loaded liposome comprising an active ingredient core, a chitosan interlayer and a sodium alginate shell layer from the inside out, wherein the active ingredient is a mixture of honokiol and rhein, and the mass ratio of honokiol to rhein is 1.5:1;

[0048] The preparation method of the colon-targeted drug-loaded liposomes comprises the following steps:

[0049] (1) adding a mixture of active ingredient, soybean lecithin and cholesterol to an organic solvent, wherein the mass ratio of active ingredient, soybean lecithin and cholesterol is 5:55:15, dissolving under heating and ultrasonic conditions to obtain a mixed solution, and then removing the organic solvent by rotary evaporation at 50° C. and 130 rpm to obtain a drug-loaded phospholipid film; adding 110 mL of PBS buffer solution with a pH of 7.4 to the drug-loaded phospholipid film to hydrate the drug-loaded phospholipid film for 10 minutes, and then ultrasonically crushing at 700 W for 3 minutes, and then filtering with a 0.45 μm filter membrane to obtain an active ingredient core solution;

[0050] (2) under stirring conditions of 400 r / min, the active ingredient core solution was added dropwise to a chitosan solution having a concentration of 0.5 mg / mL at a volume ratio of 1:1, and stirring was continued for 0.5 h after the addition was completed to obtain an active ingredient-chitosan solution;

[0051] (3) Under stirring conditions of 400 r / min, the active ingredient-chitosan solution was added dropwise to a sodium alginate solution with a concentration of 2 mg / mL at a volume ratio of 1:1, and stirring was continued for 0.5 h after the addition was completed.

[0052] Example 3

[0053] A colon-targeted drug-loaded liposome, the drug-loaded liposome comprising an active ingredient core, a chitosan interlayer and a sodium alginate shell layer from the inside out, wherein the active ingredient is a mixture of honokiol and rhein, and the mass ratio of honokiol to rhein is 0.8:1;

[0054] The preparation method of the colon-targeted drug-loaded liposomes comprises the following steps:

[0055] (1) adding a mixture of active ingredient, soybean lecithin and cholesterol to an organic solvent, wherein the mass ratio of active ingredient, soybean lecithin and cholesterol is 6:60:11, dissolving under heating and ultrasonic conditions to obtain a mixed solution, and then removing the organic solvent by rotary evaporation at 50° C. and 130 rpm to obtain a drug-loaded phospholipid film; adding 300 mL of PBS buffer solution with a pH of 7.4 to the drug-loaded phospholipid film to hydrate the drug-loaded phospholipid film for 40 minutes, and then ultrasonically crushing the film at 500 W for 12 minutes, and then filtering with a 0.45 μm filter membrane to obtain an active ingredient core solution;

[0056] (2) under stirring conditions of 400 r / min, the active ingredient core solution was added dropwise to the chitosan solution with a concentration of 4 mg / mL at a volume ratio of 1:1, and stirring was continued for 2 h after the addition was completed to obtain an active ingredient-chitosan solution;

[0057] (3) Under stirring conditions of 400 r / min, the active ingredient-chitosan solution was added dropwise to a sodium alginate solution with a concentration of 3 mg / mL at a volume ratio of 1:1, and stirring was continued for 2 hours after the addition was completed.

[0058] Example 4

[0059] A colon-targeted drug-loaded liposome, the drug-loaded liposome comprising an active ingredient core, a chitosan interlayer and a sodium alginate shell layer from the inside out, wherein the active ingredient is a mixture of honokiol and rhein, and the mass ratio of honokiol to rhein is 1.2:1;

[0060] The preparation method of the colon-targeted drug-loaded liposomes comprises the following steps:

[0061] (1) adding a mixture of active ingredient, soybean lecithin and cholesterol to an organic solvent, wherein the mass ratio of active ingredient, soybean lecithin and cholesterol is 7:75:14, dissolving under heating and ultrasonic conditions to obtain a mixed solution, and then removing the organic solvent by rotary evaporation at 50° C. and 130 rpm to obtain a drug-loaded phospholipid film; adding 260 mL of PBS buffer solution with a pH of 7.4 to the drug-loaded phospholipid film to hydrate the drug-loaded phospholipid film for 20 minutes, and then ultrasonically crushing the film at 650 W for 6 minutes, and then filtering with a 0.45 μm filter membrane to obtain an active ingredient core solution;

[0062] (2) under stirring conditions of 400 r / min, the active ingredient core solution was added dropwise to the chitosan solution with a concentration of 3 mg / mL at a volume ratio of 1:1, and stirring was continued for 1.5 h after the addition was completed to obtain an active ingredient-chitosan solution;

[0063] (3) Under stirring conditions of 400 r / min, the active ingredient-chitosan solution was added dropwise to a sodium alginate solution with a concentration of 3 mg / mL at a volume ratio of 1:1, and stirring was continued for 1.5 h after the addition was completed.

[0064] Test example

[0065] Taking the honokiol-rhein-chitosan-sodium alginate liposome (HNK / RH-CS-SALip) prepared in Example 1 as an example, the prepared HNK / RH-CS-SA Lip was subjected to drug loading, encapsulation efficiency, particle size, potential, stability and in vitro release investigations. The specific experimental methods and experimental results are as follows:

[0066] 1. Determination of drug loading and encapsulation efficiency of HNK / RH-CS-SA Lip

[0067] The encapsulation efficiency of HNK / RH-CS-SA Lip was determined by ultrafiltration centrifugation. The ultrafiltration centrifuge tube with a molecular weight cutoff of 10kd was centrifuged at 8000r / min for 30min, and the filtrate in the outer tube was taken and fixed to volume with methanol for detection.

[0068] The calculation formulas for drug loading (DL) and encapsulation efficiency (EE) are:

[0069]

[0070] W1 is the drug content of liposomes after removing free drugs, W2 is the drug content of liposome liquid without removing free drugs, and W is the total mass of feed.

[0071] After calculation, the average encapsulation efficiency of HNK / RH-CS-SA Lip was 86.38%, and the total drug loading was 6.64%.

[0072] 2. Particle size and potential determination of HNK / RH-CS-SA liposomes

[0073] The nanoparticle size distribution and Zeta potential of HNK / RH-CS-SA Lip were measured at 25°C using a nanoparticle size analyzer (Nano Brook Omni). The average particle size was 347.56±0.69nm, the PDI was 0.236±0.045, and the particle size distribution was shown in the figure. Figure 1 ; The average potential is -21.00±2.27mV.

[0074] 3. In vitro release

[0075] The drug release from the nanoparticles was evaluated by simulating the gastrointestinal pH environment using different pH media: PBS buffer (pH 1.2), PBS buffer (pH 6.8), and PBS buffer (pH 7.8).

[0076] Take 2 ml of HNK / RH-CS-SA Lip sample, put it in three dialysis bags, and then put it in 40 mL of pH 1.2, pH 6.8 and pH 7.4 medium for release. Take 0.5 mL at 0.5, 1, 2, 3, 4, 6, 8, 12, 24h and 48h, and add 0.5 mL of blank medium at the same time. The sample taken out is filtered and injected. The samples of honokiol / rhein (HNK / RH) raw material, honokiol / rhein liposome (HNK / RH Lip), honokiol / rhein / chitosan liposome (HNK / RH-CS Lip) and honokiol / rhein / chitosan / sodium alginate liposome (HNK / RH-CS-SA Lip) are operated in the same way.

[0077] like Figure 3-8 As shown, the layer-by-layer adsorption process of chitosan (CS) and sodium alginate (SA) makes the drug released less and less under the conditions of pH 1.2 and pH 6.8, and released in large quantities under the condition of pH 7.4, proving that the HNK / RH-CS-SA Lip prepared in the present application has colon targeting.

[0078] 4. Therapeutic effect of application in colitis

[0079] The mice were randomly divided into a normal group, a model group, a positive control group, a rhein raw material drug (RH) group, a honokiol raw material drug (HNK) group, a rhein / honokiol (RH / HNK) raw material drug group, and a honokiol / rhein / chitosan / sodium alginate liposome (HNK / RH-CS-SA Lip) group. Except for the normal group, mice were allowed to drink 3.5% dextran sulfate sodium (DSS) freely for 7 days to create an ulcerative colitis model in mice. After the modeling, drug liposomes were gavaged once a day for 7 consecutive days. The mice were killed after the last administration, and the colon tissue of the mice was taken to measure its length, and the content of MPO and IL6 in the tissue was detected by homogenization.

[0080] like Figure 9-11 As shown in the figure, after successful modeling, the colon length of mice was significantly shortened compared with the normal group, and the colon length was improved after oral administration, among which the HNK / RH-CS-SA Lip group had the best therapeutic effect. It was proved that the dual-drug liposome HNK / RH-CS-SA Lip had a better therapeutic effect than the drug monomer rhein API group and magnolol API group, and the combination of the two drugs had certain advantages. Compared with the combination of the two single APIs, the MPO and IL6 indicators showed that the HNK / RH-CS-SA Lip group achieved better therapeutic effects in the same treatment cycle. It may be that the modification of liposomes with chitosan and sodium alginate can delay the release time of drugs so that they can be released at a fixed point after reaching the lesion colon, which improves the overall therapeutic effect of the drug.

Claims

1. A colon-targeted drug-loaded liposome, characterized in that: The drug-loaded liposome comprises, from the inside to the outside, an active ingredient core, a chitosan interlayer and a sodium alginate shell layer.

2. The colon-targeted drug-loaded liposome according to claim 1, characterized in that: The effective ingredient is a mixture of honokiol and rhein, and the mass ratio of honokiol to rhein is 0.3-1.8:

1.

3. The method for preparing colon-targeted drug-loaded liposomes according to claim 1 or 2, characterized in that: The following steps are involved: (1) preparing an active ingredient core solution; (2) under stirring conditions, the active ingredient core solution is added dropwise to the chitosan solution, and stirring is continued for 0.5-2 hours after the addition is completed to obtain an active ingredient-chitosan solution; (3) Under stirring conditions, the active ingredient-chitosan solution is added dropwise to the sodium alginate solution, and stirring is continued for 0.5-2 hours after the addition is completed to obtain the obtained product.

4. The method for preparing colon-targeted drug-loaded liposomes according to claim 3, characterized in that: The active ingredient core solution in step (1) is prepared by the following method: (1) adding a mixture of active ingredients, phospholipids and cholesterol into an organic solvent, dissolving to obtain a mixed solution, and evaporating to remove the organic solvent to obtain a drug-loaded phospholipid film; (2) PBS buffer is added to the drug-loaded phospholipid film to hydrate the drug-loaded phospholipid film, ultrasonically disrupted, and then filtered to obtain the drug-loaded phospholipid film.

5. The method for preparing colon-targeted drug-loaded liposomes according to claim 4, characterized in that: In step (1), the mass ratio of the active ingredient, soybean lecithin and cholesterol is 5-8:50-80:10-16.

6. The method for preparing colon-targeted drug-loaded liposomes according to claim 4, characterized in that: In step (2), the volume mass ratio of PBS buffer to phospholipid is 2-5:

1.

7. The method for preparing colon-targeted drug-loaded liposomes according to claim 4, characterized in that: The hydration time in step (2) is 10-40 min.

8. The method for preparing colon-targeted drug-loaded liposomes according to claim 4, characterized in that: In step (2), the ultrasonic power is 500-700 W, and the ultrasonic time is 3-12 min.

9. The method for preparing colon-targeted drug-loaded liposomes according to claim 3, characterized in that: The concentration of the chitosan solution in step (2) is 0.4-4 mg / mL, and the volume of the active ingredient core solution is the same as that of the chitosan solution.

10. The method for preparing colon-targeted drug-loaded liposomes according to claim 3, characterized in that: The concentration of the sodium alginate solution in step (3) is 1.3-3 mg / mL, and the volume of the active ingredient-chitosan solution is the same as that of the sodium alginate solution.

Images