Oral algae motor loaded probiotic mixed delivery system and preparation method thereof

Abstract

The invention discloses a mixed delivery system of oral algae motor loaded probiotics, and relates to the technical field of biological medicine. The micron motor based on mixing of algae and probiotics can achieve the effects of rapidly reaching and being adhered to the inflammatory part of ulcerative colitis for a long time and releasing a large amount of antioxidant substances, and has high biocompatibility, high gastric acid barrier crossing performance, high adhesion targeting performance and high bioavailability. The medicine can be taken without injection, and is convenient to take and high in compliance. The preparation method of the mixed delivery system provided by the invention has the advantages of simple and rapid preparation process, mild conditions and universality, and the method has biological orthogonality. The algae-probiotic mixed delivery system is expected to provide a new strategy, a potential new drug and a drug delivery system for effectively treating intestinal diseases such as ulcerative colitis.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an oral algae motor-loaded probiotic mixed delivery system and a preparation method thereof. Background Art

[0002] Ulcerative colitis is a chronic nonspecific inflammation that mainly affects the rectum, colon mucosa and submucosa, and is an inflammatory bowel disease. According to a magazine study, the incidence of ulcerative colitis is high in Europe and the United States, and the incidence in Asia will also rise sharply. The disease is prone to recurrence, difficult to cure, and has a high risk of canceration. Patients have poor quality of life and a heavy economic burden. Traditional drug preparations have a short retention time, obvious toxic and side effects, and poor targeting. Intestinal diseases such as ulcerative colitis require drugs to overcome multiple physiological barriers to achieve efficient treatment, such as gastric acid barriers, mucus layer barriers, and intestinal cell barriers. In recent years, researchers have become increasingly aware of the correlation between reactive oxygen species, intestinal flora, and the occurrence and development of ulcerative colitis, and often use antioxidants and probiotics to treat ulcerative colitis. However, due to the poor barrier of the stomach affecting the activity of antioxidants and probiotics, and the peristalsis of the gastrointestinal tract causes the probiotics to have a short retention time in the intestine, the treatment effect of ulcerative colitis is still unsatisfactory. Therefore, there is an urgent need to develop new delivery platforms to enable probiotics and other oral antioxidants to quickly pass through the gastric barrier and retain them in the target intestinal tract for a long time, thereby effectively improving the therapeutic effect of therapeutic active substances on ulcerative colitis.

[0003] In view of this, the present invention is proposed. Summary of the invention

[0004] The purpose of the present invention is to provide an oral algae motor-loaded probiotic mixed delivery system and a preparation method thereof to achieve rapid passage of probiotics and other oral antioxidants through the gastric barrier and long-term retention in target intestinal sites, thereby effectively improving the therapeutic effect of therapeutic active substances on ulcerative colitis.

[0005] The present invention is achieved in that:

[0006] In a first aspect, the present invention provides an oral algae motor-loaded probiotic mixed delivery system, comprising an algae motor domesticated by pressure conditions and probiotics with adhesion properties, wherein the algae motor is surface-modified by a main molecule to form a first product, and the probiotics are surface-modified by a guest molecule to form a second product.

[0007] In some preferred embodiments, the algae is selected from Haematococcus pluvialis.

[0008] In some preferred embodiments, the probiotics are Escherichia coli Nissle1917, Lactobacillus rhamnosus, Bifidobacterium, Bacillus subtilis, and preferably the probiotics are Escherichia coli Nissle1917.

[0009] In some preferred embodiments, the molar ratio of the host molecule to the guest molecule is in the range of 1-100:1-100; the host molecule is cyclodextrin, cucurbituril, calixarene, pillararene or crown ether, preferably β-cyclodextrin; and the guest molecule is adamantane.

[0010] In a second aspect, the present invention also provides a method for preparing an oral algae motor-loaded mixed delivery system of probiotics, the steps of which include: incubating the first product and the second product at room temperature, and then self-assembling to obtain the mixed delivery system.

[0011] In some preferred embodiments, the first product is prepared by culturing the algae motor under pressure conditions and then modifying the surface with a main molecule, and the second product is prepared by first imparting adhesion properties to the probiotics with adhesion properties through an adhesion coating and then modifying the surface with a guest molecule.

[0012] In some preferred embodiments, the stress conditions include weak light, strong light, nitrogen deficiency, strong acid and weak acid, and the preferred stress conditions are strong light, nitrogen deficiency and weak acid environment.

[0013] In some preferred embodiments, the surface binding modification of the first product and the second product is covalent bond modification or non-covalent modification, preferably mild non-covalent modification of the host molecule and the guest molecule; the guest molecule has a good binding degree with the host molecule.

[0014] In some preferred embodiments, the adhesive coating is a polydopamine coating, a polyphenol-metal complex coating and a polysaccharide coating, preferably a polydopamine coating.

[0015] In some preferred embodiments, the room temperature incubation time is 30 minutes to 2 hours.

[0016] The present invention has the following beneficial effects:

[0017] The present invention provides an oral stress-trained algae motor loaded with adhesive probiotics mixed delivery system and a preparation method thereof. The algae motor biosynthesizes antioxidants during stress training and encapsulates them inside, and can tolerate the acidic environment of the stomach after stress training, and quickly delivers antioxidants and adhesive probiotics to the deep of the lesion through movement targeting, thereby improving the delivery efficiency of antioxidants and adhesive probiotics and reducing the premature degradation of antioxidants and adhesive probiotics when they reach the site of ulcerative colitis. Adhesive probiotics can not only regulate the intestinal microenvironment, but also solve the problem of the short residence time of algae motors in the diseased area, and improve the adhesion targeting of the delivery system. This method is used for Haematococcus pluvialis and a variety of probiotics, maintaining the structural morphology, activity and movement targeting of Haematococcus pluvialis and probiotics, maintaining the activity of antioxidants, and achieving comprehensive treatment of ulcerative colitis from multiple aspects. The oral pressure-trained algae motor-loaded mixed delivery system of adhesive probiotics proposed in the present invention fully considers the needs of patients and the disease characteristics of ulcerative colitis, and is expected to provide an innovative therapy for the targeted treatment of ulcerative colitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0019] Figure 1 The changes in biosynthesized astaxanthin of stress-trained Haematococcus pluvialis according to Example 1 of the present invention;

[0020] Figure 2 This is a scanning electron microscope image of a polydopamine coating with adhesion ability generated by polymerization on the surface of Escherichia coli Nissle1917 in Example 2 of the present invention;

[0021] Figure 3 A scanning electron microscope image of Haematococcus pluvialis and Escherichia coli Nissle1917 physically mixed or combined with a host and a guest in the oral trained hybrid microalgae motor provided in Example 3 of the present invention;

[0022] Figure 4 The motion trajectory diagrams of the untrained microalgae motor and the orally trained mixed microalgae motor provided in Example 4 of the present invention within 10 seconds after being incubated in artificial gastric juice at different time points;

[0023] Figure 5 A fluorescence image of the trained mixed microalgae motor for oral administration provided in Example 5 of the present invention to remove reactive oxygen species from intestinal epithelial cells;

[0024] Figure 6 This is an imaging diagram of the adhesion of the oral trained hybrid microalgae motor to the colon provided in Example 6 of the present invention. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0026] The following is a detailed description of an oral algae motor-loaded probiotic mixed delivery system and a preparation method thereof proposed by the present invention.

[0027] One object of the present invention is to provide an oral algae motor-loaded probiotic mixed delivery system, comprising an algae motor domesticated by pressure conditions and probiotics with adhesion properties, wherein the algae motor is surface-modified with a main molecule to form a first product, and the probiotic guest molecule is surface-modified to form a second product.

[0028] Motor carriers are micro / nanoscale device carriers that convert various energy sources into autonomous motion. On the one hand, they retain the advantages of micro-nano delivery systems, can passively or actively enrich and target inflammatory injury sites, and can effectively regulate drug release behavior. On the other hand, the excellent motion performance of motor carriers can be designed into various forms of delivery systems, overcoming the shortcomings of low barrier penetration efficiency, nonspecific adsorption and systemic toxicity of traditional micro-nanoparticles, and showing broad development prospects in the field of oral targeted drug delivery. The rapid physiological barrier penetration of motor carriers can not only avoid being metabolized by various physiological environments, but also improve the efficiency of drug delivery in gastrointestinal disease sites and reduce nonspecific adsorption of normal tissues. Although chemical propulsion and external field propulsion have shown good performance in oral delivery, their application is limited by fuel shortage, insufficient driving force, short propulsion time, easy premature degradation, necessary chemical reaction environment and complex and cumbersome external field devices.

[0029] As a rich natural renewable biological resource, algae have attracted much attention in recent years in the field of biomedicine. Due to the ease of access, easy cultivation, low cultivation cost, large surface area and rich active substances, algae have shown great application value in biomedical applications, including strong motility (>100μm / s), autofluorescence, continuous oxygen production and phototropism.

[0030] In some preferred embodiments, the algae is selected from Haematococcus pluvialis.

[0031] In some preferred embodiments, the probiotics are Escherichia coli Nissle1917, Lactobacillus rhamnosus, Bifidobacterium, Bacillus subtilis, and preferably the probiotics are Escherichia coli Nissle1917.

[0032] In some preferred embodiments, the molar ratio of the host molecule to the guest molecule is in the range of 1-100:1-100; the host molecule is cyclodextrin, cucurbituril, calixarene, pillararene or crown ether, preferably β-cyclodextrin; and the guest molecule is adamantane amine.

[0033] The guest molecule may be adamantane, ferrocene, spermine, azobenzene, etc., preferably adamantane and its related derivatives. The guest molecule needs to have a high binding constant with the host molecule in order to form a stable delivery system.

[0034] Another object of the present invention is to provide a method for preparing an oral algae motor-loaded mixed delivery system of probiotics, the steps of which include: incubating the first product and the second product at room temperature, and then self-assembling to obtain the mixed delivery system.

[0035] In some preferred embodiments, the first product is prepared by culturing the algae motor under pressure conditions and then subjecting the algae motor to surface modification with a main molecule, and the second product is prepared by first imparting adhesion properties to the adhesive probiotics through an adhesion coating and then subjecting the probiotics to surface modification with a guest molecule.

[0036] When the algae motor is subjected to stress training, a large amount of antioxidant substances will be synthesized and stored inside the algae, thereby improving the stability of the antioxidant substances and extending the half-life of the antioxidant substances in the body. The antioxidant substance is astaxanthin.

[0037] In some preferred embodiments, the stress conditions include weak light, strong light, nitrogen deficiency, strong acid and weak acid, and the preferred stress conditions are strong light, nitrogen deficiency and weak acid environment.

[0038] In some preferred embodiments, the surface binding modification of the first product and the second product is covalent bond modification or non-covalent modification, preferably mild non-covalent modification of the host molecule and the guest molecule; the guest molecule has a good binding degree with the host molecule.

[0039] The binding force between the host and guest molecules is essentially a non-covalent bond, and its strength depends on the strength of the hydrophilic and hydrophobic interaction between the host and guest molecules. The host molecule is generally a macrocyclic molecule with hydrophilic outside and hydrophobic inside, while the guest molecule is generally more hydrophobic. Typical host-guest binding pairs include cyclodextrin and adamantane, cucurbituril and ferrocene, etc. The guest molecule can easily bind to the hydrophobic cavity of the macrocyclic molecule in aqueous solution to form a stable inclusion structure.

[0040] In some preferred embodiments, the adhesive coating is a polydopamine coating, a polyphenol-metal complex coating and a polysaccharide coating, preferably a polydopamine coating.

[0041] In some preferred embodiments, the room temperature incubation time is 30 minutes to 2 hours.

[0042] Under the action of the Haematococcus pluvialis motor, the adhesive probiotics can not only quickly reach the intestines to improve the intestinal flora, but also adhere to the site of ulcerative colitis, helping the Haematococcus pluvialis to stay for a long time and release antioxidant ingredients, thereby improving the adhesion targeting of the drug.

[0043] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0044] The BBM medium and Haematococcus pluvialis (HP) used in the present invention were purchased from Shanghai Guangyu Biotechnology Co., Ltd., dopamine hydrochloride and Tris-HCl buffer were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., and Escherichia coli Nissle1917 (EcN) was purchased from Ningbo Mingzhou Biotechnology Co., Ltd. LB medium was purchased from Beijing Solebow Biotechnology Co., Ltd., β-cyclodextrin-polyethylene glycol-N-hydroxysuccinimide (CD-PEG-NHS) and adamantane-polyethylene glycol-N-hydroxysuccinimide (ADA-PEG-NHS) were purchased from Xi'an Ruixi Biotechnology Co., Ltd., NCM 460 cell line (normal human colon epithelial cells) was purchased from ATCC cell bank, and DCFH-DA fluorescent probe was purchased from Suzhou Yuheng Biotechnology Co., Ltd.

[0045] Example 1

[0046] In this embodiment, Haematococcus pluvialis is divided into two groups, one group is not subjected to stress training, and the other group is subjected to stress training under nitrogen deficiency, weak acid and strong light conditions, and then the Haematococcus pluvialis is tested under a microscope.

[0047] Normally cultured Haematococcus pluvialis was added to nitrogen-deficient, weakly acidic BBM medium and continuously illuminated for 24 hours at a light intensity of 10,000 Lux for one week. The culture solution was then collected by centrifugation, the supernatant was removed, and the induced HP was collected. The microscopic results after induction are as follows Figure 1 As shown in the figure, the left picture shows Haematococcus pluvialis without stress training, and the right picture shows Haematococcus pluvialis after stress training. The red fluorescence represents the effective ingredient, i.e. astaxanthin. Therefore, according to the microscope results, it can be seen that Haematococcus pluvialis can only synthesize astaxanthin in large quantities after stress training.

[0048] Example 2

[0049] In this example, E. coli is divided into two groups, one group is not subjected to modification operation on E. coli, and the other group is subjected to modification of E. coli using polydopamine coating.

[0050] After culturing EcN in LB medium for 4 hours, it was washed with PBS, added to Tris-HCl buffer (pH 8.8, 1mM) containing dopamine hydrochloride, and cultured at room temperature for 0.5 hours. The polydopamine-coated EcN (EcN@PDA) was collected by centrifugation (8000r / min), washed 3 times with PBS, and purified to obtain EcN@PDA. The scanning electron micrograph is shown in Figure 2 As shown, Figure A shows the Escherichia coli Nissle1917 without any modification, and Figure B shows the Escherichia coli Nissle1917 after polydopamine coating.

[0051] Example 3

[0052] This example prepares an oral algae motor-loaded probiotic mixed delivery system.

[0053] First, the normally cultured Haematococcus pluvialis was added to the nitrogen-deficient, weakly acidic BBM medium and illuminated continuously for 24 hours at a light intensity of 10,000 Lux for one week. The culture medium was then collected by centrifugation, the supernatant was removed, and the induced HP was collected.

[0054] Then, EcN was cultured in LB medium for 4 hours and washed with PBS. EcN was added to Tris-HCl buffer (pH 8.8, 1 mM) containing dopamine hydrochloride and cultured at room temperature with shaking for 0.5 hours. The polydopamine-coated EcN (EcN@PDA) was collected by centrifugation (8000 r / min), washed three times with PBS, and purified to obtain EcN@PDA.

[0055] Secondly, the induced HP was placed in a PBS solution, treated with 10 μM CD-PEG-NHS at room temperature for 2 hours, and the excess CD-PEG-NHS was washed away to obtain CD-HP, which was dispersed in PBS.

[0056] Again, EcN@PDA was added to PBS solution and treated with 10 μM ADA-PEG-NHS for 2 h at room temperature. After washing away the excess ADA-PEG-NHS, ADA-EcN was obtained and dispersed in PBS.

[0057] Finally, excess ADA-EcN was added to CD-HP and incubated for 2 h. Unbound ADA-EcN was removed by low-speed centrifugation at 1000 rpm to obtain EcN-HP hybrid micromotors.

[0058] The final result is Figure 3 It can be seen that, among them, I is a single Haematococcus pluvialis, II is a mixed incubation of Haematococcus pluvialis without β-cyclodextrin modification and probiotics without adamantane (ADA) modification, III is a mixed incubation of Haematococcus pluvialis without β-cyclodextrin modification and probiotics modified with ADA, IV is a mixed incubation of Haematococcus pluvialis modified with β-cyclodextrin and probiotics without ADA modification, and V is a mixed incubation of Haematococcus pluvialis modified with β-cyclodextrin and probiotics modified with ADA. In the figure, the red signal is Cy5-labeled Haematococcus pluvialis, and the green signal is GFP-labeled Escherichia coli Nissle1917, and the scale is 10μm. This result shows that HP and EcN can only be combined through host-guest interaction after being modified with the host molecule and the guest molecule respectively.

[0059] Example 4

[0060] In this example, a group of untrained Haematococcus pluvialis motors and a group of EcN-HP hybrid micromotors prepared in Example 4 were selected. The two groups were incubated in artificial gastric juice (pH 3.5) for different periods of time, and the movement trajectories of the two motors were recorded for 10 seconds using a microscope. The results are shown in FIG. Figure 4 shown.

[0061] According to the results, the untrained Haematococcus pluvialis motor quickly lost its motility in artificial gastric juice, while the EcN-HP hybrid micromotor still maintained good motility after being in contact with artificial gastric juice for 2 hours. This is sufficient to show that the EcN-HP hybrid micromotor prepared according to the technical solution of this application has a good performance in resisting gastric acid.

[0062] Example 5

[0063] In this example, the EcN-HP hybrid micromotor prepared in Example 4 and 300 μM hydrogen peroxide were incubated with NCM460 cells for 12 h, and then the cells were incubated in a medium containing 10 μM DCFH-DA for 30 min and then washed with PBS. Finally, the DCF fluorescence image in the cells was obtained by laser confocal microscopy. The results are shown in Figure 2. Figure 5 As shown, according to the test results, the EcN-HP hybrid micromotor has the best ability to effectively remove reactive oxygen species from cells.

[0064] Example 6

[0065] In this example, three experimental groups were set, namely, trained Haematococcus pluvialis motors (HP), EcN+HP physical mixed group, and EcN-HP hybrid micromotors. The three groups were incubated with mouse colon in vitro at 37°C for 1 hour, and then imaged using a live animal imaging device. The results are shown in FIG. Figure 6According to the results, the EcN-HP hybrid micromotor combines with EcN with adhesion ability through supramolecular action, so that the hybrid motor can stay in the colon better, improve its retention time, and further exert the therapeutic effect of probiotics and Haematococcus pluvialis.

[0066] In summary, the EcN-HP hybrid micromotor prepared in this embodiment has good gastric acid resistance, so that it can maintain its motility in the acidic environment of the stomach, overcome multiple physiological barriers, and reduce nonspecific adsorption and removal of non-target sites. In addition, the micromotor carries antioxidant substances, which can not only effectively remove reactive oxygen in cells, but also regulate the oxidation state in cells through various mechanisms. At the same time, the micromotor contains probiotics with adhesion properties, which can not only regulate the intestinal microenvironment, but also solve the problem of short residence time of algae motors in diseased areas, thereby improving the adhesion targeting of the delivery system.

[0067] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An oral algae motor-loaded probiotic mixed delivery system, characterized in that: It includes an algae motor domesticated by pressure conditions and a probiotic with adhesion properties. The algae motor forms a first product after surface modification by a main molecule, and the probiotic forms a second product after surface modification by a guest molecule.

2. The oral algae motor-loaded probiotic mixed delivery system according to claim 1, characterized in that: The algae is selected from Haematococcus pluvialis.

3. The oral algae motor-loaded probiotic mixed delivery system according to claim 1, characterized in that: The probiotics are Escherichia coli Nissle1917, Lactobacillus rhamnosus, Bifidobacterium, Bacillus subtilis, and preferably the probiotics are Escherichia coli Nissle1917.

4. The oral algae motor-loaded probiotic mixed delivery system according to claim 1, characterized in that: The molar ratio of the host molecule to the guest molecule is in the range of 1-100:1-100; the host molecule is cyclodextrin, cucurbituril, calixarene, pillararene or crown ether, preferably β-cyclodextrin; and the guest molecule is adamantane.

5. A method for preparing the oral algae motor-probiotic mixed delivery system as described in any one of claims 1 to 4, characterized in that: The steps include: the first product and the second product are incubated at room temperature and then self-assembled to obtain the mixed delivery system.

6. The method for preparing an oral algae motor-in-probiotic mixed delivery system according to claim 5, characterized in that: The first product is prepared by culturing the algae motor under pressure conditions and then modifying the surface of the main molecule. The second product is prepared by first giving the adhesive probiotics adhesive properties through an adhesive coating and then modifying the surface of the probiotics with guest molecules.

7. The method for preparing an oral algae motor-in-probiotic mixed delivery system according to claim 6, characterized in that: The stress conditions include weak light, strong light, nitrogen deficiency, strong acid and weak acid, and the preferred stress conditions are strong light, nitrogen deficiency and weak acid environment.

8. The method for preparing an oral algae motor-in-probiotic mixed delivery system according to claim 6, characterized in that: The surface binding modification of the first product and the second product is covalent bond modification or non-covalent modification, preferably mild non-covalent modification of the host molecule and the guest molecule; the guest molecule has a good binding degree with the host molecule.

9. The method for preparing an oral algae motor-probiotic mixed delivery system according to claim 6, characterized in that: The adhesive coating is a polydopamine coating, a polyphenol-metal complex coating and a polysaccharide coating, preferably a polydopamine coating.

10. The method for preparing an oral algae motor-in-probiotic mixed delivery system according to claim 5, characterized in that: The room temperature incubation time is 30 minutes to 2 hours.

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