CN114533764A - Lipid membrane coated probiotic as well as preparation and application thereof - Google Patents
Lipid membrane coated probiotic as well as preparation and application thereof Download PDFInfo
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- CN114533764A CN114533764A CN202111679133.2A CN202111679133A CN114533764A CN 114533764 A CN114533764 A CN 114533764A CN 202111679133 A CN202111679133 A CN 202111679133A CN 114533764 A CN114533764 A CN 114533764A
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- A61K35/741—Probiotics
- A61K35/742—Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a lipid membrane coated probiotic, and a preparation method and an application thereof. The invention uses self-assembled biological membrane to pack bacteria, thus obtaining a probiotic bacteria packed by lipid membrane; the probiotic preparation can enhance the viability of bacteria in gastrointestinal fluids, increase the colonization of bacteria in intestinal tracts and the like, and can be used as an enhancement therapy for preventing and treating tumors.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to lipid membrane coated probiotics and preparation and application thereof.
Background
The existing research shows that the bacteria in the gastrointestinal tract play a vital role in the occurrence and development of various diseases, including cancer, obesity, diabetes, depression and the like, so that the bacteria prepared into a gastric retention dosage form has great development prospect for treating the diseases.
There is increasing evidence that gut microbiota can be used to enhance the anti-tumor efficacy of cancer immunotherapy. Commensal bacteria have been found to promote the development and progression of malignancies by affecting both the local and systemic immune systems. The deregulation of the gut microbiome has been shown to affect the therapeutic efficacy of PD-1 blockers in tumor-bearing mice and cancer patients, whereas oral supplementation with specific strains can improve the anti-tumor immune response. One potential way that the microflora may modulate the vaccine response is to provide natural adjuvants that enhance the vaccine response, but oral or other direct administration of probiotics results in poor probiotic bioavailability, inadequate intestinal residence time or reduced immunogenicity of the bacteria, and also an inability to avoid phagocytosis and clearance of the bacteria by the reticuloendothelial system, which leads to poor therapeutic efficacy of the probiotic.
Disclosure of Invention
The invention aims to solve the problems and provides lipid membrane coated probiotics and preparation and application thereof.
According to the technical scheme of the invention, the lipid membrane is coated with probiotics and comprises probiotics and a lipid membrane coating the probiotics, wherein the lipid membrane comprises anionic lipid, neutral lipid and cholesterol.
The lipid membrane of the present invention modifies the surface of the probiotic bacteria and encapsulates it within the lipid membrane, thereby protecting against the adverse environment of the gastrointestinal tract and avoiding immune system clearance.
Further, the probiotic bacteria are selected from one or more of cocci, bacilli, spirochetes and actinomycetes.
Specifically, the probiotic bacteria are selected from one or more of yeast (such as Saccharomyces cerevisiae, Saccharomyces uvarum, and Saccharomyces rouxii), probiotic bacillus (such as Bacillus subtilis and Bacillus licheniformis), Clostridium butyricum, Lactobacillus (such as Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus jensenii, and Lactobacillus raman), coccus (such as Streptococcus faecalis, lactococcus lactis, and Streptococcus intermedius), Bifidobacterium (such as Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium bifidum, Bifidobacterium adolescentis, Bifidobacterium ovoicus, and Bifidobacterium thermophilum), and actinomycetes.
Preferably, the probiotic is lactobacillus rhamnosus and/or bifidobacterium longum.
The anionic lipid is selected from DOPG (dioleoylphosphatidylglycerol) and/or DPPG (dipalmitoylphosphatidylglycerol); the neutral lipid is selected from DPPC (dipalmitoylphosphatidylcholine) and/or DOPE (dioleoylphosphatidylethanolamine)
Further, the molar ratio of the anionic lipid to the neutral lipid to the cholesterol is 3-4: 16-18: 4-6.
Preferably, the anionic lipid is DOPG and the neutral lipid is DPPC; molar ratio of DOPG, and cholesterol 17: 83: 25.
the second aspect of the present invention provides a method for preparing the above lipid membrane coated probiotic, comprising the steps of,
s1: suspending probiotic culture with OD600 value of 0.6-0.8 in buffer solution containing cation to obtain bacterial solution;
dissolving anionic lipid, neutral lipid and cholesterol in organic solvent, and drying to obtain lipid membrane;
s2: and adding the lipid membrane into the bacterial solution to prepare the lipid membrane coated probiotic.
Further, the concentration of the cation in the buffer is 10-15mM, and the buffer containing the cation may be a PBS solution (4 ℃) containing calcium chloride and/or sodium chloride.
Further, the total mass of anionic lipid, neutral lipid and cholesterol added is 0.01-2g per mL of probiotic culture (OD600 value is 0.6-0.8); preferably, it is 0.1 to 0.2 g.
Further, the organic solvent is dichloromethane and/or trichloromethane.
Specifically, probiotic cultures were grown overnight in MRS medium by growing the probiotic bacteria at 37 ℃; the overnight cultures were diluted to fresh MRS medium and grown for 6h at 37 ℃; the bacteria were collected by centrifugation at 4200rpm for 10 min and resuspended in ice-cold PBS. Bacterial concentrations were determined by Nanodrop 2000.
15mL of probiotic culture were washed and suspended in 10mL of ice-cold PBS solution containing 12.5mM calcium chloride; DOPG, DPPC (molar ratio 17:83) and cholesterol were dissolved in 5mL of dichloromethane at a molar ratio of 4: 1; subjecting the obtained solution to rotary evaporation by using a rotary evaporator, and drying the lipid film at room temperature (25 + -5 deg.C); hydrating the obtained lipid membrane in 10mL of bacterial solution, vortexing for 15min, and preparing the lipid membrane-coated probiotic by utilizing electrostatic adsorption.
In a third aspect of the invention, there is provided a sustained release formulation comprising the lipid film-coated probiotic as described above.
Further, the administration mode of the sustained-release preparation is one or more of oral administration, intraperitoneal injection, subcutaneous injection, intravenous injection and intramuscular injection, and oral administration and intratumoral injection are preferred.
The fourth aspect of the invention provides the application of the lipid membrane coated probiotic or the sustained-release preparation in preparing a medicament for preventing and/or treating tumors.
Further, the tumor is colon cancer or carcinoma in situ.
Compared with the prior art, the technical scheme of the invention has the following advantages: coating the bacteria with a self-assembled biofilm to obtain a lipid-coated probiotic; the probiotic preparation can enhance the viability of bacteria in gastrointestinal fluids, increase the colonization of bacteria in intestinal tracts and the like, and can be used as an enhancement therapy for preventing and treating tumors.
Drawings
Fig. 1 is a schematic view of the method for coating probiotic bacteria with lipid membrane in example 1.
Fig. 2 is a TEM image of lipid membrane coated probiotics of example 2.
Fig. 3 is a graph of particle size of lipid membrane coated probiotics in example 2.
FIG. 4 is a confocal laser microscopy image of lipid membrane-coated bacteria of example 2.
FIG. 5 is a plot of the growth (left) and viability of the lipid membrane coated bacteria of example 2 (right).
FIG. 6 is a flow analysis plot of the growth of lipid membrane coated probiotics of example 2 in simulated gastric fluid, simulated intestinal fluid, PBS.
Fig. 7 shows the survival of the lipid membrane coated probiotic bacteria and the lipid membrane uncoated probiotic bacteria in the intestine of example 2 after 8 h.
FIG. 8 shows the tumor volume, survival and weight change of mice in the prevention experiment of lipid membrane-coated probiotics and non-lipid membrane-coated probiotics in example 3.
FIG. 9 shows the tumor volume, survival and weight changes of mice in the prevention experiment of oral lipid membrane-coated probiotics and non-lipid membrane-coated probiotics in example 4.
FIG. 10 is a graph showing the change in survival time of mice during induction of orthotopic colon cancer in mice in example 5.
FIG. 11 is a graph showing the body weight changes of mice during the induction of orthotopic colon cancer in the mice in example 5.
FIG. 12 is a graph of H & E staining of mouse colon during induction of orthotopic colon cancer in mice in example 5.
FIG. 13 is a graph of AB/APS staining of mouse colon during induction of orthotopic colon cancer in mice in example 5.
FIG. 14 shows the tumor volume, survival time and body weight change of mice in the treatment experiment of lipid membrane-coated probiotic and lipid membrane-uncoated probiotic prepared by intratumoral injection in example 6.
Detailed Description
The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.
Example 1 preparation of lipid film coated probiotic
Lactobacillus rhamnosus (ATCC7469) was grown overnight at 37 ℃ in MRS medium. Overnight cultures were diluted to fresh MRS medium and grown at 37 ℃ for 6 h. The bacteria were harvested by centrifugation at 4200rpm for 10 min and resuspended in ice-cold PBS. Bacterial concentrations were determined by Nanodrop 2000.
The preparation method of the lipid membrane entrapping bacteria comprises the following steps: 15mL of the bacterial culture was washed and suspended in 10mL of a 12.5mM ice-cold PBS solution containing calcium chloride. Mixing a molar ratio of 17:83 DOPG ((0.38mg) and DPPC (1.66mg) were dissolved in 5mL dichloromethane at a molar ratio of 4:1 with cholesterol (0.27mg), a FITC-labeled coating was prepared by co-assembly with FITC-mPEG2000-DSPE (molar ratio of FITC-mPEG2000-DSPE to DOPG and DPPC 10%, 0.86mg), the resulting solution was spin evaporated using a rotary evaporator, the lipid film was dried at room temperature, the resulting lipid film was hydrated in 10mL bacterial solution and vortexed for 15min and then stored at 4 ℃ for further characterization, fig. 1 is a schematic of the preparation process.
Other bacteria coated with lipid membranes can be prepared by the same method.
Example 2 characterization of lipid membrane coated probiotics
The lipid membrane-coated bacterial structures were visualized using transmission electron microscopy (HT7700, Hitachi). A drop of the prepared bacterial formulation solution was deposited on a carbon-coated copper grid. Then dried in an oven at 60 ℃ overnight. And drying under a tungsten lamp for 5min on the next day, and observing. Transmission Electron Microscope (TEM) images (fig. 2) show that Lipid membrane coated bacteria (hereinafter LCB) prepared from lactobacillus rhamnosus (ATCC7469) have a distinct outer shell on the surface, in sharp contrast to the sharp edges of unencapsulated lactobacillus rhamnosus (ATCC 7469). Dynamic Light Scanning (DLS) measurements (fig. 3) showed an increase in both size and zeta potential after encapsulation of the lipid membrane.
The encapsulation of bacteria was examined using a confocal laser confocal microscope using a lipid membrane containing FITC (fluorescein isothiocyanate) green fluorescent label (FITC labeled lipid membrane was prepared by co-assembling a mixed lipid material with FITC-mPEG2000-DSPE (10% molar ratio and DOPG DPPC)) and E.coli expressing RFP red fluorescence. RFP expressing bacteria are red in color, lipid membranes containing FITC are green in color, and they appear yellow after co-localization. The results are shown in FIG. 4, indicating successful lipid membrane coating.
To determine whether the coating of lipid membranes would have an effect on the survival and viability of probiotic bacteria, the growth and viability of E.coli expressing RFP red fluorescence was determined using a cytometry kit-8 (CCK-8). As shown in fig. 5, there was little difference in probiotic viability and viability between the encapsulated and unencapsulated lipid membranes in LB medium.
Survival of LCB was further assessed in simulated gastric fluid supplemented with pepsin (SGF, pH 1.2) and simulated intestinal fluid containing trypsin (SIF, pH 6.8). As shown in fig. 6, LCB showed significantly improved tolerance to simulated gastric fluid (left) and simulated intestinal fluid (middle) compared to probiotic bacteria that were not coated with lipid membrane.
To verify whether lipid membranes could enhance in vivo activity, mice were gavaged with 1X 108The colonization of the gastrointestinal tract is assessed after CFU coating with lipid membranes or without lipid membrane-coated probiotics. The presence of the probiotic was imaged using an In Vivo Imaging System (IVIS) 4h, 48h after administration. As shown in the results of fig. 7, it is shown that the survival and colonization of the encapsulated bacteria in the gastrointestinal tract are increased.
According to the report that lactobacillus rhamnosus and bifidobacterium have the effect of treating tumors, in order to evaluate whether the probiotics coated by the lipid membrane can be used as an enhanced therapy for preventing and treating tumors, the effects of two bacteria coated by the lipid membrane in preventing and treating tumors are researched.
Example 3 oral prevention of colon cancer by lipid membrane-coated probiotic
In colon cancer prevention experiments, C57/BL6 mice (6-8 weeks) were used to construct MC38 colon cancer tumor models and were bred under Specific Pathogen Free (SPF) conditions. Mice were randomized into 3 groups: PBS group, two naked bacterium oral group and two dosage form bacterium oral group.
Oral preparation containing 1 × 10 of two naked bacteria (Lactobacillus rhamnosus ATCC7469, Bifidobacterium longum GDMCC1.1258)8CFU Lactobacillus rhamnosus and 1X 108A suspension of 150. mu.L of Bifidobacterium longum in bacterial suspension of CFU was intragastrically administered to mice once every seven days for five times.
The two-dosage form of oral preparation is prepared from a composition containing 1 × 108CFU Lactobacillus rhamnosus dosage forms and 1 × 108The mice were gavaged with a 150 μ LPBS bacterial suspension of CFU bifidobacterium longum formulation once every seven days for five times. After the administration is finished, the composition will contain 2X 106100 μ LPBS of individual tumor cells was injected subcutaneously into the right flank of the mice.
Body weight monitoring was performed every other week during the dosing period; tumor size and body weight were measured every three days after tumor cell inoculation, and tumor volume in mice was measured as 0.52 × a × b2(tumors were measured every three days by digital vernier calipers, with a major diameter and b major diameter).
Waiting until the length of the tumor of the mouse exceeds 20mm or the volume of the tumor exceeds 2000mm3At that time, the mice were taken out of the SPF animal house and sacrificed. Tumor volume size (left), survival (middle) and body weight recordings (right) are shown in fig. 10.
Example 4 lipid film-coated probiotic subcutaneous injection to prevent colon cancer
In colon cancer prevention experiments, C57/BL6 mice (6-8 weeks) were used to construct MC38 colon cancer tumor models and were bred under Specific Pathogen Free (SPF) conditions. Mice were randomly divided into PBS group, two naked bacteria subcutaneous injection group, and two formulation bacteria subcutaneous injection group.
30mL of Lactobacillus rhamnosus and Bifidobacterium longum cultures were washed and suspended in 15mL of 12.5mM CaCl2Was prepared from the strain in ice-cold PBS solution and autoclaved for 20 min.
Two naked bacterium subcutaneous injection groups were prepared by mixing the mixture containing inactivated 1X 108CFU RhamnusLactobacillus sacchari and 1X 108 A 50 μ LPBS bacterial suspension of CFU bifidobacterium longum was injected subcutaneously into mice once every seven days for a total of five injections.
The two-dosage form subcutaneous injection group is used for preparing bacterial antigen of the bacterial strain by autoclaving for 20min to prepare the bacterial antigen, and then the dosage form of the bacterial antigen is prepared according to the method. After the preparation is finished, the catalyst will contain 1X 108CFU Lactobacillus rhamnosus dosage forms and 1 × 108A 50 μ LPBS bacterial suspension of CFU bifidobacterium longum formulation was injected subcutaneously into mice once every seven days for a total of five injections. After the administration is finished, the composition will contain 2X 106100 μ LPBS of individual tumor cells was injected subcutaneously into the right flank of the mice.
Body weight monitoring was performed every other week during dosing; tumor size and body weight were measured every three days after tumor cell inoculation, and tumor volume in mice was measured as 0.52 × a × b2(tumors were measured every three days by digital vernier calipers, with a major diameter and b major diameter). Waiting until the tumor length of the mouse exceeds 20mm or the volume exceeds 2000mm3At time, mice were taken out of the SPF animal house for sacrifice. Tumor volume size (left), survival (middle) and weight recordings (right) are shown in fig. 9.
Example 5 lipid membrane-coated probiotic prevention of in situ colon cancer
In orthotopic colon cancer prevention experiments, C57/BL6 mice (6-8 weeks) were used to construct MC38 colon cancer tumor models, and were bred in Specific Pathogen Free (SPF) conditions. Mice were randomized into 3 groups: PBS group, two naked bacterium oral group and two dosage form bacterium oral group.
The in situ PBS group was administered in the same manner as the subcutaneous group. The in situ two naked bacterium oral administration group is prepared from 1 × 108CFU Lactobacillus rhamnosus and 1X 108A suspension of CFU bifidobacterium longum in 150 μ LPBS was intragastrically administered to mice once every seven days for a total of five intragastrically administered doses.
The in situ two-dosage form of the oral composition is prepared from 1 × 108CFU Lactobacillus rhamnosus dosage forms and 1 × 108The mice were gavaged with a 150 μ LPBS bacterial suspension of CFU bifidobacterium longum formulation once every seven days for a total of five times.
In the experiment for preventing colon cancer in situ, after the administration of each group of mice is finished, in-situ induction and tumor formation are carried out. C57/BL6 mice (6-8 weeks) were used to construct orthotopic colon cancer tumor models and were bred under Specific Pathogen Free (SPF) conditions. AOM/DSS was used for in situ induction of colon cancer in C57/BL6 mice. Mice of 6-8 weeks of age were selected, intraperitoneally injected with azomethane AOM (10mg/kg) once the first day, fed drinking water for 2% DSS for 5 days the third day, weighed daily to assess response to DSS-induced colon cancer, then fed with regular drinking water for 2 weeks, and weighed every 3 days. Wherein 2% DSS and conventional drinking water feeding are circulated for three times, the survival time result of the mice detected in the induction process is shown in figure 10, and the weight result is shown in figure 11.
After the induction was completed to 80 days, the mice were sacrificed and the colons were removed, histologically, immunohistochemically and microscopically fresh colons were fixed with 4% paraformaldehyde for 24H, further processed and H & E stained. Alcian blue and periodic acid schiff staining (AB/PAS) revealed goblet cells according to the manufacturer's instructions (supplied by novice). After applying DAB pigment carrier, the tissue sections were subjected to hematoxylin staining, dehydration, fixation, H & E staining and AB/APS staining experimental results are shown in FIGS. 12 and 13.
EXAMPLE 6 lipid Membrane-coated probiotic intratumoral injection for treatment of colon cancer
In therapeutic experiments, C57/BL6 mice (6-8 weeks) were used to construct MC38 colon cancer tumor models and were raised under Specific Pathogen Free (SPF) conditions. After the MC38 mouse colon cancer cells were passaged, the tumor cells were collected and resuspended in PBS buffer to contain 2X 10 cells 6100 μ LPBS of individual tumor cells was injected subcutaneously into the right flank of the mice. Three days later, mice were randomized into 3 groups according to tumor size: PBS group, two naked bacterium intratumoral injection group and two preparation type bacterium intratumoral injection group.
The two groups injected with naked bacterium in tumor are used for preparing tumor according to the above-mentioned method, and the fourth day of tumor preparation contains 1X 108CFU Lactobacillus rhamnosus and 1X 108 A 50 μ LPBS bacterial suspension of CFU bifidobacterium longum was injected into the mice tumors once every other day for a total of two weeks.
Two-dosage form intratumoral injection groupThe fourth day after the above process would contain 1X 108CFU Lactobacillus rhamnosus dosage form and 1 × 108A 50 μ LPBS bacterial suspension of CFU bifidobacterium longum formulation was injected into the mice tumors once every other day for two weeks (formulation preparation method described above).
After dosing was complete, mice continued to monitor tumor volume (left), survival (middle) and body weight (right), as shown in FIG. 14, until mice had tumors that exceeded 20mm in length or had a volume of over 2000mm3At that time, the mice were brought out of the animal house and sacrificed.
In conclusion, the probiotic preparation can enhance the viability of bacteria in gastrointestinal fluids, increase the colonization of bacteria in intestinal tracts and the like, and can be used as an enhancement therapy for preventing and treating tumors.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Various other modifications and alterations will occur to those skilled in the art upon reading the foregoing description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the spirit or scope of the invention.
Claims (10)
1. A lipid membrane-coated probiotic comprising a probiotic and a lipid membrane coating said probiotic, said lipid membrane comprising anionic lipids, neutral lipids and cholesterol.
2. The lipid film coated probiotic according to claim 1, wherein the probiotic is selected from one or more of cocci, bacilli, spirochetes and actinomycetes.
3. The lipid film coated probiotic according to claim 1, wherein the anionic lipid is selected from DOPG and/or DPPG; the neutral lipid is selected from DPPC and/or DOPE.
4. The lipid membrane-coated probiotic according to claim 1 or 3, characterized in that the molar ratio of anionic lipids, neutral lipids and cholesterol is from 3 to 4: 16-18: 4-6.
5. A method for preparing a lipid membrane coated probiotic according to any of claims 1 to 4, comprising the steps of,
s1: suspending the probiotic culture in a buffer solution containing cations to obtain a bacterial solution;
dissolving anionic lipid, neutral lipid and cholesterol in organic solvent, and drying to obtain lipid membrane;
s2: and adding the lipid membrane into the bacterial solution to prepare the lipid membrane coated probiotic.
6. The method according to claim 5, wherein the concentration of the cation in the buffer is 10 to 15 mM.
7. The method according to claim 5, wherein the organic solvent is dichloromethane and/or chloroform.
8. A sustained-release preparation comprising the lipid film-coated probiotic according to any one of claims 1 to 4.
9. Use of a lipid membrane coated probiotic according to any one of claims 1 to 4 or a sustained release formulation according to claim 7 in the manufacture of a medicament for the prevention and/or treatment of a tumour.
10. The use of claim 9, wherein the tumor is colon cancer or carcinoma in situ.
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