Preparation method and application of probiotic-drug oral co-delivery system

Abstract

The invention discloses a preparation method and application of a probiotic-drug oral co-delivery system, a method of coating probiotics with phospholipid and coating indissolvable drugs with cyclodextrin is adopted, so that the stability and activity of the probiotics in an acid environment of gastric acid are ensured, the damage of gastric juice to the probiotics is effectively reduced, and the bioavailability of the oral co-delivery system is improved. Enough viable bacteria quantity and vigor reach the intestinal tract are ensured, the stability of the treatment effect is ensured, the problem that the expected effect cannot be exerted due to insufficient viable bacteria quantity is avoided, and the infection risks such as acute gastroenteritis and urethritis possibly caused by excessive viable bacteria quantity are prevented; the traditional Chinese medicine composition has good adaptability, can immediately exert a treatment effect without dormancy adjustment, can rapidly and effectively supplement probiotics, is safe and harmless to human bodies, does not generate toxic or side effects after being taken, is simple and convenient in preparation method, is easy to operate, is suitable for industrial large-scale production, and has a wide application prospect. An efficient and safe new scheme is provided for treating intestinal inflammation through co-delivery of probiotics and chemical drugs.

Description

Technical Field

[0001] The present invention relates to the technical fields of probiotics and chemical drug delivery, and in particular to a preparation method and application of a probiotic-chemical drug co-delivery system. Background Art

[0002] Inflammatory bowel disease (IBD) is a non-specific chronic inflammatory disease characterized by long-term inflammation of the colon and small intestine, mainly ulcerative colitis and Crohn's disease. Many studies have shown that the disease is closely related to intestinal mucosal immune system dysfunction and gut microbiota dysbiosis. Clinically, corticosteroids, antibiotics, aminosalicylates, immunosuppressants, and biologics are used for clinical intervention in IBD, which can relieve symptoms to a certain extent; however, long-term use of these drugs can lead to serious complications, such as opportunistic infections, malignancies, autoimmunity, and hepatotoxicity. Therefore, there is an urgent need to develop effective and safe treatment methods for IBD.

[0003] Escherichia coli, which is commonly present in the lower part of the small intestine of warm-blooded animals and is harmless to the host, is a normal flora in the animal intestine. The optimal growth temperature of Escherichia coli is 37 °C, and the pH range for its survival is 4.0 to 9.0. When the pH is too low, its survival ability will be affected. When orally administering live Escherichia coli or related preparations, they must pass through gastric juice, and the pH of gastric juice ranges from 0.9 to 1.5, which will cause a large number of live bacteria to inactivate, thus significantly reducing the taking effect.

[0004] Azathioprine, as an immunosuppressant, has significant anti-inflammatory effects. However, azathioprine has extremely low solubility in water, which directly affects its bioavailability in the human body. It is reported in the literature that the oral absorption rate of azathioprine is 50%, and this relatively low absorption rate may be partly attributed to its poor solubility in water. Therefore, when clinically using azathioprine, there is an urgent need to improve its solubility. Summary of the Invention

[0005] The present invention provides a preparation method of an oral co-delivery system of probiotics and drugs to solve the problems of unstable taking effect and potential safety hazards caused by the active degradation of probiotics during taking, the uncertain number and vitality of probiotics reaching the intestine, and the low absorption rate of chemical drugs.

[0006] To achieve the above object, the technical solutions provided by the present invention are as follows:

[0007] A preparation method of an oral co-delivery system of probiotics and drugs comprises the following steps:

[0008] (1) Cultivate the probiotics to 10 8 -10 9Centrifuge after reaching CFU / mL, and resuspend with PBS;

[0009] (2) Fuse 0.01 - 0.04 mmol of phospholipid and 0.002 - 0.08 mmol of cholesterol in 2 mL of chloroform, and rotary evaporate at room temperature until a uniform thin film state is reached, then dry at 37 °C to obtain a liposome membrane;

[0010] (3) Take 1 mL of the resuspended bacterial solution from step (1) and mix it with the liposome membrane, stir for 10 - 30 min to obtain a bacterial solution coated with liposomes; (4) Mix 60 - 150 mg of DSPE-PEG-COOH, 10 - 30 mg of EDC, and 4 - 8 mL of H2O evenly until it becomes transparent; Mix 26.7 - 76.7 mg of NH2-β-CD, 10 - 30 mg of NHS, and 6 mL of H2O, then heat in a water bath until it becomes transparent; Mix the above two transparent solutions, stir for 24 h, dialyze for 2 days, and freeze-dry to obtain DSPE-PEG-β-CD;

[0011] (5) Add the drug to ammonia water with a mass concentration of 10%, dissolve DSPE-PEG-β-CD in H2O, mix the two and stir for 2 h, rotary evaporate at 35 °C to dryness, add to H2O, centrifuge, and take the supernatant for freeze-drying to obtain a DSPE-PEG-β-CD inclusion complex; (6) Incubate the liposome-coated bacterial solution with the DSPE-PEG-β-CD inclusion complex at 37 °C, and then freeze-dry to obtain an oral co-delivery system of probiotics - drugs.

[0012] The probiotic in step (1) is a probiotic with the effect of alleviating intestinal inflammation, specifically including Escherichia coli Nissle1917, etc.

[0013] The centrifugation in step (1) is carried out at 5000 rpm - 10000 rpm for 5 - 30 min.

[0014] In step (5), 15 - 40 mg of the drug is added to 1 mL of ammonia water with a mass concentration of 10%.

[0015] In step (5), 60 - 150 mg of DSPE-PEG-β-CD is dissolved in 5.4 mL of H2O.

[0016] The drug in step (5) is a poorly soluble chemical drug with the effect of treating intestinal inflammation, specifically including azathioprine, etc.

[0017] Application of the oral co-delivery system of probiotic-drug prepared by the preparation method of the present invention in drugs for treating intestinal inflammation. The present invention characterized the experimental results of bacteria encapsulated in liposomes by methods such as laser confocal microscopy and flow cytometry. Through cell experiments, its in vitro anti-inflammatory properties, cytotoxicity experiments and laser confocal microscopy were used to observe the biocompatibility of the system and its effects on cell migration and cell growth. Also, by measuring and analyzing the body weight changes of mice, pathological sections and staining of colon tissues and spleen tissues, and observing indicators such as spleen organ index, the effects of the co-delivery system on protecting the intestinal barrier function and colon tissue structure were evaluated.

[0018] Advantages of the present invention:

[0019] (1) The combination of probiotics and chemical drugs for treating diseases in the present invention is a new treatment strategy, which shows great potential especially in the field of treating inflammatory bowel disease. By regulating the intestinal microbiota, the treatment effect can be enhanced and its side effects can be reduced. The application of the probiotic-chemical drug co-delivery system has multiple advantages. Firstly, it can provide a high concentration of local drug, thereby prolonging the pharmacological activity of the drug and maximizing the drug efficacy. Secondly, the co-delivery system can prevent or reduce the degradation and potency loss of the drug before it reaches the site of action.

[0020] (2) The probiotic-chemical drug co-delivery system proposed by the present invention uses phospholipid inclusion technology to protect probiotics, effectively reducing their loss in the gastric acid environment and preventing the inhibition of probiotics by chemical drugs, ensuring that probiotics reach the intestine in sufficient quantity and activity, thereby enhancing the treatment effect. This system precisely regulates the number of viable bacteria reaching the intestine, avoiding the risk of ineffectiveness due to too few viable bacteria or infections such as acute gastroenteritis and urethritis caused by too many viable bacteria, ensuring the safety and effectiveness of the treatment.

[0021] (3) The probiotic-chemical drug co-delivery system proposed by the present invention uses materials harmless to the human body such as DSPE-PEG-β-CD and phospholipids, and has no toxicity or side effects after administration. Its preparation process is simple and easy to implement, and is suitable for large-scale industrial production. Description of the drawings

[0022] Figure 1 It is the infrared spectrum of DSPE-PEG-β-CD;

[0023] Figure 2 It is the nuclear magnetic resonance spectrum of DSPE-PEG-β-CD;

[0024] Figure 3 It is the laser confocal image of phospholipid-encapsulated Escherichia coli Nissle 1917;

[0025] Figure 4Flow analysis diagram of phospholipid - encapsulated Escherichia coli Nissle 1917;

[0026] Figure 5 For Escherichia coli Nissle 1917 cultured to 10 8 -10 9 CFU / mL, comparison diagram of absorbance between freeze - dried powder and normal bacterial solution;

[0027] Figure 6 Effect of β - CD@azathioprine on the viability of macrophage RAW264.7;

[0028] Figure 7 Effect of β - CD@azathioprine on the morphology of macrophage RAW264.7;

[0029] Figure 8 Effect of β - CD@azathioprine on the secretion of nitric oxide (NO) by macrophage RAW264.7;

[0030] Figure 9 Change diagram of body weight of dextran sulfate sodium - induced colitis mice by Escherichia coli Nissle 1917 - DSPE - PEG - β - CD@azathioprine;

[0031] Figure 10 Digital photograph of colon length of colitis mice by Escherichia coli Nissle 1917 - DSPE - PEG - β - CD@azathioprine;

[0032] Figure 11 Analysis diagram of spleen index of colitis mice by Escherichia coli Nissle 1917 - DSPE - PEG - β - CD@azathioprine;

[0033] Figure 12 Effect diagram of Escherichia coli Nissle 1917 - DSPE - PEG - β - CD@azathioprine on the mRNA expression of intestinal inflammatory factors in colitis mice. Detailed implementation mode

[0034] The technical solutions of the present invention will be further described in detail below through examples. However, the content of the present invention is not limited thereto. In the examples, the methods are conventional methods unless otherwise specified, and the materials, reagents, etc. are prepared from commercial sources or by conventional methods that have been publicly disclosed unless otherwise specified.

[0035] The phospholipids used in the examples are egg yolk lecithin and 1,2 - dioleoyl - sn - glycero - 3 - phosphoric acid sodium salt (DOPA).

[0036] Example 1

[0037] Preparation method of Escherichia coli Nissle 1917-DSPE-PEG-β-CD@azathioprine, the specific steps are as follows:

[0038] (1) Culture Escherichia coli Nissle 1917 to 10 8 -10 9 CFU / mL (at 37 °C for about 6 h), centrifuge at 8000 rpm for 8 min, take 1 mL of the bacterial solution and resuspend it with 1 mL of PBS to obtain a resuspended bacterial solution;

[0039] (2) Dissolve 0.02 mmol of phospholipid (egg yolk lecithin) and 0.005 mmol of cholesterol in 2 mL of chloroform, use a 25 mL brown bottle, rotary evaporate at room temperature until a uniform thin film state is obtained, and dry at 37 °C to obtain a liposome membrane;

[0040] (3) Take 1 mL of the resuspended bacterial solution and mix it with the liposome membrane in step (2), stir for 15 min to obtain a liposome-coated bacterial solution;

[0041] (4) Mix 100 mg of DSPE-PEG-COOH, 20 mg of EDC, and 6 mL of H2O and stir magnetically until it becomes transparent (500 rpm, 30 °C) to obtain a DSPE-PEG-COOH mixed solution; mix 56.7 mg of NH2-β-CD, 20 mg of NHS, and 6 mL of H2O, and stir magnetically until it becomes transparent (500 rpm, 30 °C) to obtain an NH2-β-CD mixed solution; mix the DSPE-PEG-COOH mixed solution with the NH2-β-CD mixed solution, stir at 25 °C for 24 h, dialyze with a dialysis bag with a molecular weight cut-off of 2500 for 2 days, and freeze-dry to obtain DSPE-PEG-β-CD;

[0042] (5) Dissolve 25 mg of the drug azathioprine in 1 mL of ammonia water with a mass concentration of 10%, dissolve 100 mg of DSPE-PEG-β-CD in 5.4 mL of H2O, mix the two, stir at 30 °C for 2 h, spin dry, add 2 mL of H2O, centrifuge, and take the supernatant for freeze-drying to obtain a DSPE-PEG-β-CD inclusion complex;

[0043] (6) Incubate the liposome-coated bacterial solution in step (3) and the DSPE-PEG-β-CD inclusion complex obtained in step (5) together at 37 °C for 1.5 h, and freeze-dry for 48 h to obtain Escherichia coli Nissle 1917-DSPE-PEG-β-CD@azathioprine, weigh it, and calculate the yield to be 59.43%.

[0044] In step (2), 0.002 mmol of nile red was also added to stain the material, and other process conditions and raw materials were the same as those in Example 1 to prepare stained Escherichia coli Nissle 1917-DSPE-PEG-β-CD@azathioprine.

[0045] Example 2

[0046] Characterization of the Escherichia coli Nissle 1917-DSPE-PEG-β-CD@azathioprine co-delivery system:

[0047] 1. Infrared spectrum

[0048] The infrared spectrum data was recorded in the spectral range of 4000 cm -1 to 500 cm -1 by using a total reflection Fourier infrared spectrometer, and the characteristic functional groups were analyzed. As Figure 1 shown in the infrared spectrum of DSPE-PEG-β-CD, the connection of DSPE-PEG-COOH and NH2-β-CD mainly involves the formation of amide bonds. The N-H stretching vibration of the amide bond appears at 3423 cm -1 , which is a broad and strong absorption peak. The C=O stretching vibration of the amide bond appears at 1735 cm -1 , which is a strong absorption peak.

[0049] 2. Nuclear magnetic resonance spectrum

[0050] Weigh 20 mg of the freeze-dried DSPE-PEG-β-CD product, dissolve it in 600 μL of d6-DMSO, and use a fully digital nuclear magnetic resonance spectrometer to detect its structure. Determine its structure by 1H NMR. As Figure 2 shown in the nuclear magnetic resonance spectrum of DSPE-PEG-β-CD, the obvious characteristic peaks of β-CD, PEG, and DSPE appear in the spectrum, indicating successful synthesis.

[0051] 3. Laser confocal spectrum

[0052] By staining the bacteria with calcein-AM, it can penetrate the live cell membrane. Through the action of esterase, the almost non-fluorescent Calcein-AM is de-esterified to generate the green fluorescent substance Calcein with strong fluorescence signal (Ex / Em: 495 nm / 515 nm). Therefore, the green fluorescence can be detected. By setting the experimental groups of bacteria stained and membrane not stained (bacteria stained), bacteria not stained and membrane stained (membrane stained), and bacteria stained and membrane stained (all), as Figure 3 shown, it indicates that the overlap degree of nile red and calcein-AM is relatively high, and it can be basically determined that the inclusion is successful.

[0053] 4. Flow Cytometry Atlas

[0054] Take 10 8 -10 9 CFU / mL of encapsulated bacterial solution. By setting up two experimental groups: bacteria not stained and membrane not stained, and bacteria not stained and membrane stained. The membrane was stained with Nile-Red dye. Using a flow cytometer, the absorption wavelength was detected at 528 nm. As Figure 4 shown, the results indicate that compared with uncoated bacteria, the fluorescence intensity of the experimental group coated with Escherichia coli increased significantly, demonstrating the presence of the coating membrane.

[0055] 5. Comparison Chart of Absorbance between Lyophilized Bacterial Solution and Normal Bacterial Solution

[0056] Culture Escherichia coli Nissle1917 in LB medium until 10 8 -10 9 CFU / mL (at 37°C for about 6 h). Then centrifuge 1 mL of the bacterial solution (8000 rpm, 8 min), discard the supernatant, resuspend with 10% skim milk powder, and then freeze-dry to obtain the lyophilized powder, i.e., the bacterial powder (unactivated); Add one tube of the lyophilized powder to 20 ml of LB medium and activate for 6.5 h to obtain the bacterial solution of the activated bacterial powder (bacterial powder (activated)). As Figure 5 shown, the absorbance of the original bacterial solution, the bacterial powder (unactivated), and the bacterial solution of the activated bacterial powder (bacterial powder (activated) in the figure) was compared at 600 nm, indicating that the activity of the prepared bacterial powder after activation is basically the same as that of the original bacterial solution.

[0057] 6. In Vitro Activity Test

[0058] For the drug absorption and release in the DSPE-PEG-β-CD@azathioprine system, due to the poor water solubility of azathioprine, a low dose of DMSO was used as the solvent. In the experiment, a DMSO experimental group was designed to exclude the influence of the solvent on the anti-inflammatory effect of RAW264.7 cells. Figure 6 The experimental results show that when the DMSO concentration is 100 - 400 μg / mL, the anti-inflammatory effect on RAW264.7 cells is the most obvious. Figure 7 And Figure 8 respectively show the effects of DSPE-PEG-β-CD@azathioprine on the morphology and secretion of nitric oxide (NO) of macrophages RAW264.7. The results indicate that DSPE-PEG-β-CD@azathioprine has the effect of maintaining the normal cell morphology of inflammatory cells, and the effect of the azathioprine group (XD50, AZA, 40 mg / kg) is stronger than that of the pure bacteria group (P50, 200 mg / kg, 10 8 -10 9CFU / mL, EcN1917) was more obvious, and after treatment with DSPE-PEG-β-CD@azathioprine group (P100, 40 mg / kg), the relative expression of NO was closer to the expression level of normal cells, and there was also a significant difference from the model group (LPS), proving that this system has the effect of repairing colitis cells.

[0059] 7. In vivo activity test

[0060] The method for treating dextran sulfate sodium (DSS)-induced colitis model with Escherichia coli Nissle1917-DSPE-PEG-β-CD@azathioprine is as follows:

[0061] The mice were randomly divided into 6 groups (n = 5 / group): blank group (Con), model group (DSS), pure bacteria group (P50, 200 mg / kg, 10 8 -10 9 CFU / mL, EcN1917), azathioprine group (XD50, AZA, 40 mg / kg), DSPE-PEG-β-CD@azathioprine group (XD25, 40 mg / kg, product of step 5, colonies and cells cannot be cultured simultaneously), Escherichia coli Nissle1917-DSPE-PEG-β-CD@azathioprine group (P100, 40 mg / kg). Except for the normal control group, the other groups were given sterile drinking water containing 3% dextran sulfate sodium DSS to drink freely for 7 days, and then replaced with pure drinking water without dextran sulfate sodium DSS. During the modeling, drug treatment was given. Escherichia coli Nissle1917-DSPE-PEG-β-CD@azathioprine or 5-aminosalicylic acid (positive drug) was suspended in 1 mL of PBS solution and gavaged once a day for 8 consecutive days.

[0062] Figure 9 The figure shows the changes in the body weight of mice in each group treated with drugs for the colitis model. It can be observed that the changes in the blank group (Con) and the model group (DSS) are extremely obvious. The body weight of the mice in the blank group (Con) increases with time, while the body weight of the mice in the model group (DSS) decreases with time. This is because of the effects such as loss of appetite and bloody stools caused by the modeling, which confirms the success of the modeling. The body weight of the mice in the experimental groups (XD25, XD50, P50, P100) is between the blank group (Con) and the model group (DSS), and the effect of maintaining body weight in the experimental group (P100) is close to that of the blank group (Con), indicating that Escherichia coli

[0063] Nissle1917-DSPE-PEG-β-CD@azathioprine has the effect of maintaining the body weight change of mice.

[0064] Figure 10Digital photographs of the colon length during the treatment of colitis model mice with each group of drugs. As can be seen in the bar graph, the comparison between the blank group (Con) and the model group (DSS) is extremely significant, with three stars; while the comparison between the pure bacteria group (P50) and the model group (DSS) is not very obvious because the action cycle of probiotics is relatively long. Therefore, it is not very obvious. The other experimental groups (XD25, XD50, P100) have relatively obvious differences compared with the model group (DSS), and the gap from the blank group (Con) is not very large. Therefore, it can be judged that Escherichia coli Nissle1917-DSPE-PEG-β-CD@azathioprine has the effect of alleviating intestinal inflammation in mice.

[0065] Figure 11 Statistical analysis chart of the spleen weight of colitis model mice treated with each group of drugs. The data shows that the comparison between the blank group (Con) and the model group (DSS) is extremely significant. It can be seen that the spleen index of the pure bacteria group (P50) is relatively close to that of the model group (DSS), which is because the action cycle of probiotics is relatively long, so it is not very obvious. However, the spleen index also shows a certain decrease, indicating that probiotics have a alleviating effect; while the action effects of the experimental groups (XD25, XD50, P100) are relatively obvious, and the spleen index of the experimental group (P100) is closest to that of the blank group (Con), indicating that Nissle1917-DSPE-PEG-β-CD@azathioprine can alleviate the impact of intestinal inflammation on the spleen of mice.

[0066] Figure 12 Effects of each group of drugs on the mRNA expression of intestinal inflammatory factors in colitis mice. Compared with the DSS group, the mRNA expressions of pro-inflammatory cytokines IL-1β, IL-6 and TNF-α in the colon tissues of mice in the experimental groups (XD25, XD50, P50, P100) and the positive group decreased, while the expression of the anti-inflammatory cytokine IL-10 increased significantly.

[0067] The above results indicate that Escherichia coli Nissle1917-DSPE-PEG-β-CD@azathioprine can effectively improve systemic inflammation and repair colon damage without affecting the growth and weight changes of mice.

[0068] The specific embodiments of the present invention have been described in detail above in conjunction with the accompanying drawings. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those of ordinary skill in the art, various changes can be made without departing from the purpose of the present invention.

Claims

1. A method for preparing a probiotic-drug oral co-delivery system, characterized in that: Here are the steps: (1) Cultivate the probiotics to 10 8 -10 9 CFU / mL and then centrifuged and resuspended in PBS; (2) 0.01-0.04 mmol phospholipid and 0.002-0.08 mmol cholesterol were mixed in 2 mL chloroform, evaporated at room temperature until a uniform film was formed, and dried at 37°C to obtain a liposome membrane; (3) 1 mL of the resuspended bacterial solution in step (1) was mixed with the liposome membrane and stirred for 10-30 min to obtain a liposome-coated bacterial solution; (4) 60-150 mg DSPE-PEG-COOH, 10-30 mg EDC, and 4-8 mL H2O were mixed evenly until transparent; 26.7-76.7 mg NH2-β-CD, 10-30 mg NHS, and 6 mL H2O were mixed and then incubated in a water bath until transparent; the above two transparent solutions were mixed, stirred for 24 h, dialyzed for 2 days, and freeze-dried to obtain DSPE-PEG-β-CD; (5) adding the drug to 10% ammonia water, dissolving DSPE-PEG-β-CD in H2O, mixing and stirring the two for 2 h, spinning and drying, adding H2O, centrifuging, and freeze-drying the supernatant to obtain DSPE-PEG-β-CD inclusion complex; (6) The liposome-encapsulated bacterial solution was incubated with the DSPE-PEG-β-CD inclusion complex at 37°C and freeze-dried to obtain the probiotic-drug oral co-delivery system.

2. The method for preparing the oral co-delivery system of probiotics and drugs according to claim 1, characterized in that: The probiotics in step (1) are probiotics that can relieve intestinal inflammation.

3. The method for preparing the oral co-delivery system of probiotics and drugs according to claim 1, characterized in that: The centrifugation in step (1) is carried out at 5000 rpm-10000 rpm for 5-30 min.

4. The method for preparing the oral co-delivery system of probiotics and drugs according to claim 1, characterized in that: Step (5) 15-40 mg of the drug is added to 1 mL of 10% ammonia water.

5. The method for preparing the oral co-delivery system of probiotics and drugs according to claim 1, characterized in that: Step (5) 60-150 mg of DSPE-PEG-β-CD was dissolved in 5.4 mL of H2O.

6. The method for preparing the oral co-delivery system of probiotics and drugs according to claim 1, characterized in that: The drug in step (5) is a chemical drug that is poorly soluble and has the effect of treating intestinal inflammation.

7. Use of the probiotic-drug oral co-delivery system prepared by the method of claim 1 in drugs for treating intestinal inflammation.

Images