CN111996148A - Surface-modified microorganism, and preparation method and application thereof - Google Patents
Surface-modified microorganism, and preparation method and application thereof Download PDFInfo
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- CN111996148A CN111996148A CN202010910772.4A CN202010910772A CN111996148A CN 111996148 A CN111996148 A CN 111996148A CN 202010910772 A CN202010910772 A CN 202010910772A CN 111996148 A CN111996148 A CN 111996148A
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Abstract
The present invention relates to a method for preparing a surface-modified microorganism having a surface with a coating, comprising the steps of treating bacteria with a salt solution to allow the bacteria surface to adsorb cations; the anionic polymer is added to form a coating on the surface of the bacteria through self-assembly, so that the bacteria are protected from being eroded by gastric acid, bile acid and digestive enzyme, and the oral availability of the bacteria is greatly improved; meanwhile, the wrapped bacteria are inactivated temporarily, and the surrounding environment response de-coating can be intelligently identified, so that the in-vivo selection and targeted delivery of the bacteria are realized.
Description
Technical Field
The invention relates to the technical field of biology, and relates to a surface-modified bacterium, a preparation method and application thereof, in particular to a surface-coated bacterium, a preparation method and application thereof.
Background
Naturally, various microbial communities colonize the human body, inhabit our body and establish a complex ecosystem to influence physiological homeostasis and host function. As reported, the pathogenesis, disease progression and treatment of many diseases, such as inflammatory bowel disease, multiple sclerosis, diabetes, allergy, asthma, autism and cancer, are believed to be influenced by the composition of the microbial community. Because bacteria can interact with their niches in the human body and can also produce some key biomolecules, they respond to a variety of diseases for diagnostic and therapeutic purposes. Bacterial communities residing in different tissues of the human body always play a special role in maintaining human health. Probiotics, which are viable microorganisms beneficial to the host, are very important tools for microbial management, and can be used in the treatment and diagnosis of diseases, to regulate the immune system, cellular metabolism, and epithelial barrier function, among others. However, not all probiotics are beneficial in all cases and there are still many challenges for clinical transformation when developing therapies targeting microbiota, developing technologies to enhance efficacy and providing methods of targeted delivery to specific tissues to ensure biocompatibility and therapeutic efficacy are critical.
The intestine is the largest reservoir of microorganisms in the human body. The gut flora affects many aspects of host physiology and is considered to be a key factor in mediating host fitness maintenance. Recent findings in microbiology highlight the importance of the Gastrointestinal (GI) microbiome in regulating human health and disease, including cancer, obesity, diabetes, clostridium difficile, and depression. Thus, the delivery of probiotics to the gut to affect and modulate gut microbial composition may potentially affect the treatment of many human diseases. Oral administration of probiotics is considered convenient and well accepted and patient compliance. However, for oral probiotic bacteria, the problem encountered by probiotics is to be able to overcome the acidity and enzymes present in the stomach, an environment which has been shown to affect the activity and therapeutic effectiveness of the probiotic bacteria. The fatal drawback of current commercial probiotic products is that the insufficient delivery of probiotic bacteria to the lower part of the gastrointestinal tract, especially the acidic environment of the stomach, limits the conversion of many probiotic delivery technologies during oral delivery. Due to the limited efficacy of current use of probiotics by oral delivery, the ability of probiotics to protect them from acids and enzymes in the stomach and to target delivery to the gut for the well-defined, health-promoting purpose would be of great value. To date, microencapsulation has been defined as the primary technique to address the problem of oral administration of probiotics to prevent direct contact of the probiotic with the harsh environment of the gastric tract. Unfortunately, this method also prevents direct interaction of the probiotics with the surface of the intestine, thereby reducing their colonization in the intestine. In particular, the over-introduction of microencapsulated chemicals can also produce a range of side effects on the biological activity of the probiotic and on the health of the host. Therefore, this approach would not be of sufficient advantage in animal models or human transformation.
Disclosure of Invention
In view of the above-mentioned problems of the prior art, the inventors have provided surface-modified bacteria and a method for preparing them, in particular bacteria having a surface film (of polymer) which ensure the temporary inactivation of the bacteria (e.g. probiotics) in the stomach, making it possible to overcome the damage of acids and enzymes present in the stomach; while being delivered to the intestine, the encapsulated bacteria automatically remove their coating in the intestine and restore their activity as needed, with the purpose of selectively releasing bacteria, particularly probiotics, to the targeted lower digestive tract, depending on the low pH and digestive enzymes in the digestive tract which cause denaturation.
The present invention relates, in one aspect, to a method for preparing a surface-modified bacterium having a surface coating thereon, comprising the steps of,
treating the bacteria with a salt solution to allow the bacteria surface to adsorb cations,
adding a polymer which can act with cation static electricity to form a coating on the surface of the bacteria by self-assembly.
Preferably, the present invention relates to a method for preparing a surface-modified bacterium, comprising the steps of:
treatment of bacteria with calcium salt solutions to adsorb Ca on the surface of the bacteria2+Ions;
polymers are added to self-assemble on the bacterial surface to form a coating.
Preferably, the bacteria are treated in suspension with a calcium phosphate solution containing calcium chloride to allow Ca to be adsorbed on the surface of the bacteria2+Ions;
the anionic polymer solution is added and then the pH is lowered to enable complete self-assembly on the bacterial surface to form a coating.
Preferably, the pH reduction is equal to or less than 5; further preferably, the anionic polymer is preferably an anionic polymer L100-55.
In another aspect, the present invention relates to a surface-modified bacterium having a surface with an envelope, wherein the envelope comprises:
a polymer; and the combination of (a) and (b),
calcium ions for crosslinking the anionic polymer.
Preferably, the polymer is a pH-responsive polymer, an enzyme-responsive polymer, a light-responsive polymer, a reductive-responsive polymer; preferably, the polymer is a pH-responsive anionic polymer, preferably the pH-responsive anionic polymer is capable of dissolving at a pH >5.5, preferably the pH-responsive anionic polymer is capable of dissolving at a pH > 6. After being wrapped, the bacteria are temporarily inactivated when the pH is less than 5.5, the environment can be intelligently identified, and when the pH is more than 5.5, the bacteria can be quickly revived after the outer membrane is removed in a response manner, so that the bacteria can be controlled to be released and targeted delivery can be realized;
preferably, the pH-responsive anionic polymer is an anionic polymer L100-55.
The invention also relates to a method for improving the structure of the intestinal flora, which can resist the erosion of gastric acid and digestive enzyme by oral administration or gastrointestinal administration of the surface modified bacteria, so as to inhibit the growth and/or colonization of intestinal pathogenic bacteria and selectively increase the content and oral availability of certain bacteria in the intestinal tract; preferably, the bacteria are selected from the group consisting of probiotics, preferably one or more combinations of escherichia coli, enterobacter faecalis, staphylococcus aureus, probiotic bacillus, clostridium butyricum, lactobacillus, bifidobacterium and actinomycetes.
Preferably, the method for improving the intestinal flora structure further comprises oral or intestinal administration of an antibacterial drug.
The invention also relates to a method for treating intestinal inflammation and/or improving intestinal function, characterized in that the surface-modified bacteria are administered orally or gastrointestinal to inhibit the growth of intestinal pathogens and/or to eliminate pathogens.
Drawings
FIG. 1a is a schematic diagram of a method for producing a bacterium having an envelope according to the present invention;
FIGS. 1b-c are transmission electron microscope images of bacteria having envelopes according to the present invention;
fig. 1d is a transmission electron microscope image of bacteria with an envelope formed in PBS at pH 7.4 to which the present invention relates;
FIG. 1e is an image of a laser scanning confocal microscope of bacteria with an envelope according to an embodiment of the present invention;
FIG. 1f is a graph showing the growth of bacteria having an envelope according to an embodiment of the present invention;
FIG. 1g shows the change in zeta potential of bacteria with an envelope according to an embodiment of the present invention;
FIG. 2 is a transmission electron microscope image of bacteria having different bacterial thicknesses in an embodiment of the invention;
FIGS. 3a-b are schematic illustrations of the temporary inactivation of encapsulated bacteria at lower pH conditions in an embodiment of the present invention;
FIGS. 3c-d are graphs showing the on-demand rejuvenation of encapsulated bacteria at higher pH conditions in accordance with an embodiment of the present invention;
FIGS. 3e and 3f illustrate the removal of the coating from the bacterial surface in accordance with an embodiment of the present invention;
FIG. 3g is a confocal microscope image of SIF incubated with membrane detached from the bacterial surface;
FIG. 3h shows a variation of bacteria with an envelope in the gastrointestinal tract according to an embodiment of the present invention;
FIG. 4a is a graph showing the tolerance of bacteria having a coating film to simulated gastric fluid in an embodiment of the present invention;
FIG. 4b shows the growth of EcN and bacteria with an envelope (EcN-L) in intestinal fluid (SIF) in this example of the invention;
FIGS. 4c-4h show the survival of bacteria with an envelope (EcN-L) and bacteria without an envelope EcN in the stomach and intestine of a mouse according to an embodiment of the present invention;
FIG. 4i shows the survival rates of bacteria with an envelope (EcN-L) and bacteria without an envelope EcN in the stomach and intestine of mice according to an embodiment of the present invention;
FIG. 4j is an image of bacteria with an envelope (EcN-L) and non-envelope from the gastrointestinal tract ex vivo in an embodiment of the invention;
FIG. 5a is a graph of EcN retention with a thicker capsule in an example embodiment of the invention;
FIGS. 5b-5e are viability assays of EcN-L with thicker envelopes and EcN without envelopes in embodiments of the invention;
FIGS. 6a-6c are graphs showing the survival of K88 when EcN and bacteria EcN-L with an envelope were incubated with K88 in an example embodiment of the present invention;
FIG. 6d is a graph of the number of K88 in feces from mice fed EcN-L and EcN in a specific example of the invention;
FIGS. 6e-6f are counts of K88 after collection of intestinal contents and tissues after sacrifice of mice according to embodiments of the invention;
FIGS. 6g and 6h are graphs showing the effect of bacteria with a coating on the colonization of K88 in the intestine according to an embodiment of the present invention.
Detailed Description
The present application will be described in further detail with reference to the accompanying drawings and embodiments. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention. It should be noted that, for convenience of description, only the portions related to the invention are shown in the drawings. Other parts which are not explicitly shown or described are understood as conventional means or solutions of the prior art, which may be combined with the technical features shown in the present invention to achieve the technical effects of the present invention.
It should be noted that, in the case of no conflict, specific additional technical features in the embodiments and examples of the present invention may be combined with or replaced with each other. The present application will be described in detail below with reference to the embodiments with reference to the attached drawings.
The surface-modified bacteria of the present invention may be coated with exogenous natural or non-natural membranes. The present invention relates to a surface-modified bacterium having a surface coated with a polymer. In a preferred embodiment of the present invention, the surface-modified bacteria of the present invention have a surface coating formed by an anionic polymer (preferably L100-55), wherein the surface of the bacteria is first adsorbed with cations, and then the anionic polymer is electrostatically reacted with the cations on the surface of the bacteria to form a self-contained coating, for example, in a specific embodiment of the present invention, the bacteria are treated with a calcium salt solution (e.g., calcium chloride solution) to make the surface of the bacteria adsorb Ca2+Ions, followed by the addition of anionic polymers to self-assemble on the bacterial surface to form a coating. As shown in the schematic flow chart of FIG. 1a, Ca is adsorbed on the surface of bacteria2+Ionic, after addition of anionic polymer, the pH is lowered to enable anionic polymer with Ca2+Are electrostatically interacted with each other atThe surface of the bacteria self-assembles to form a coating. In a particular embodiment of the invention, the bacteria are treated (e.g., suspended) with a calcium phosphate solution comprising calcium chloride. The inventors have found that the integrity of the coating is superior when the pH is lowered by less than or equal to 5 (relative to an environment where the pH is above 5, e.g. 7.4 in a particular embodiment).
The bacteria may be probiotic bacteria or bacteria species for medical use. In this embodiment, the bacteria may be one or more of escherichia coli, enterococcus faecalis, bacillus subtilis, clostridium butyricum, lactobacillus, bifidobacterium, and actinomycetes; for example, Escherichia coli Nissel 1917(E.coli Nissel 1917, EcN), Bacillus subtilis, Enterococcus faecalis (Enterococcus faecalis), Staphylococcus aureus (Staphylococcus aureus).
The bacterial surface has a coating, formed by the method of manufacture described with particular reference to the above or particular examples, comprising a polymer, calcium ions, wherein the calcium ions are used to crosslink the anionic polymer. In a preferred embodiment of the invention, the polymer is an anionic polymer (e.g., L100-55); in a preferred embodiment, the anionic polymer is a pH-responsive anionic polymer that can decompose or disintegrate at or below or above a particular pH. The bacterial coating according to the present invention can be dissolved, disintegrated or disintegrated at pH >5.5 or pH >6, for example, at pH 6.8, and further, the release of bacteria and the targeted delivery of bacteria can be controlled, for example, bacteria can be disintegrated and disintegrated again in the intestinal tract without being decomposed by gastric acid to protect the survival of bacteria, and bacteria can be released. In other embodiments of the present invention, the polymer forming the envelope may be an enzyme-responsive polymer, a light-responsive polymer, or a reductively-responsive polymer. The enzyme responsive polymer degrades in the presence of certain enzymes (such as certain digestive enzymes, hydrolytic enzymes, etc.), the photoresponsive polymer can degrade or degrade in the presence of light, and the reducibility responsive polymer can degrade or degrade in the presence of a reducing enzyme or reducing agent, and thus can degrade at a specific location (in the intestinal tract). The surface modified bacteria of the invention can be used for coating at pH <5.5, temporarily inactivating the bacteria and recognizing the environment, and can be quickly compounded after being removed from the outer membrane in response when the pH is greater than 5.5, so that the bacteria can be controlled to release and targeted delivery can be realized.
According to the characteristics of the bacteria with the coating film, the bacteria can be used for improving the intestinal flora structure, for example, the method comprises the steps that the bacteria with the surface modification (namely the bacteria with the coating film) are orally taken or gastrointestinal tract administered, the bacteria can resist the erosion of gastric acid and digestive enzyme, and can be released and proliferated in the intestinal tract, so that the growth and colonization of pathogenic bacteria in the intestinal tract are inhibited, the number of pathogenic bacteria is reduced, the content and the oral availability of beneficial bacteria (probiotics) in the intestinal tract are increased, and the intestinal flora structure can be improved finally. Wherein the bacteria are selectively increased, and are selected from Escherichia coli, enterococcus faecalis, Staphylococcus aureus, probiotic bacillus, Clostridium butyricum, Lactobacillus, Bifidobacterium, and Actinomycetes, or a combination thereof. The method for improving the structure of the intestinal flora according to the conventional practice of the current medical treatment may be administered after oral or enteral administration of an antibacterial agent, with the above surface-modified bacteria. The antibacterial drug can be natural drug or extract thereof, such as berberis thunbergii, coptis chinensis and the like, or antibiotic, such as quinolone, beta-lactam, macrolide and the like. In addition, the bacteria may be used to treat intestinal inflammation and/or improve intestinal function by orally or parenterally administering the bacteria to inhibit the growth of intestinal pathogens and/or to eliminate pathogens, which may be used in combination with an antibacterial agent.
The invention will be further illustrated and explained with reference to the following specific examples.
Experimental materials and strains
Coli e.coli Nissle 1917 (hereinafter EcN), and e.coli K88 (hereinafter K88) were purchased from the chinese common collection of microorganisms and cell cultures (GMCC). Plasmids pBBR1MCS2-Tac-mCherry (kanamycin resistance) and pMP-2444-GFP (gentamicin resistance, GFP expression). Commercial Luria-Bertani (LB) was used as the cloning medium, and other reagents were purchased from domestic suppliers and used as received.
Bacteria with envelope
Example 1
Anionic Polymer L100-55 (purchased from Evonik, Piscataway, N.J.) was dissolved in PBS solution at a concentration of 4mg/ml, and 0.5ml of the bacterial culture was washed and resuspended in 1ml of a solution containing 12.5mM CaCl2To the ice-cold calcium phosphate solution, then the appropriate L100-55 solution is added. After mixing for a few minutes, the pH of the resulting solution is lowered to
Coli EcN (designated EcN-L) having an envelope (coating), i.e., a bacterium having an envelope, was obtained by resuspension by centrifugation (using dilute hydrochloric acid) and washed in PBS (pH 5). As shown in FIG. 1a, Ca2+The ions adsorb to the surface of EcN due to the negative charge of their surface, converting the surface of EcN to a positive charge to form cross-links. Then, based on the anionic polymers L100-55 with Ca2+Electrostatic interaction between the two, when the PBS pH is higher<5.5 at the decreased level, the anionic polymer L100-55 self-assembled at EcN.
The morphology of EcN-L was observed using a transmission electron microscope (HITACH, Japan). EcN-L was dispersed in suitable water and then deposited onto a carbon coated copper mesh. The samples were then completely dried in air prior to observation. Then, L100-55 and Green Fluorescent Protein (GFP) expressed in EcN were labeled with Cy3 and further visualized by laser scanning confocal microscopy (Leica TCS SP8, germany) EcN-L and EcN. The average size and apparent Zeta potential of EcN-L and EcN were determined by dynamic light scattering (Malvern Zetasizer nano ZS, UK).
Transmission electron microscopy images show EcN surface covered with a thin and transparent outer layer (envelope), as shown in fig. 1b and 1c, indicating that a small amount of polymer can achieve complete coverage for the bacteria. As shown in FIG. 2, by varying the amount of L100-55 (20 μ L L100-55 solution in FIG. 2a and 30 μ L L100-55 solution in FIG. 2 b), the thickness of the coating on the surface of the bacteria is also varied, i.e., the thickness of the coating assembled on the surface of the bacteria can be effectively adjusted.
The integrity of the envelope depends on the pH of the solution, e.g. PBS used for storage and resuspension, and the integrity of the bacterial envelope formed in PBS at pH 7.4 as shown in figure 1d is worse than at pH 5 (shown in figure 1 b). L100-55 was labeled with the dye Cy3 and EcN-L was further visualized by Laser Scanning Confocal Microscopy (LSCM). As shown in FIG. 1e, GFP (green) expressing EcN was quantitatively enveloped by L100-55 (red). Dynamic Light Scanning (DLS) measurements showed that EcN increased in size after encapsulation of L100-55, but the zeta potential decreased from-15.6 mV to-17 mV (FIG. 1g), indicating the presence of anionic carboxyl groups of L100-55 on the EcN surface. To examine whether the wrapped envelope (polymer layer) had an effect on the viability of EcN, a growth curve was recorded (fig. 1 f). The growth rate of coated EcN was slightly retarded within the first 4 hours compared to uncoated EcN, which should be due to the fact that the incompletely removed coating on the surface of the probiotic bacteria limited its growth. Subsequently, EcN-L reached the same rate of OD600 growth at 5h as uncoated EcN, indicating that the coating was completely removed and that the effect on bacterial viability after coating EcN with L100-55 was negligible.
Example 2
Encapsulated EcN was tested for reactivation on demand, controlled release, and pH response stability. As shown in fig. 3a-b, when the encapsulated bacteria were cultured in LB at pH 5 at 37 ℃, the bacteria were difficult to grow and showed temporary inactivation; when cultured in LB at pH 7 (FIGS. 3c-d) at 37 ℃, the bacteria rapidly proliferate as they come out of the membrane and exhibit the property of reviving as needed. As shown in TEM images (fig. 3e), when EcN encapsulating the envelope was incubated in SIF at pH 6.8, behavior of the envelope from swelling to rupture and finally complete removal from EcN surface was observed, which process took about 4 hours. The length of time indicated that the coated ECN showed a strong potential in controlling EcN release into the target intestine and that the bacterial morphology remained intact after SIF exposure. The response of the encapsulated EcN in SIF was also monitored by flow cytometry (fig. 3f) for the detachment process.
The pH response stability of the envelope (anionic polymer) of envelope-encapsulated EcN in SIF was examined using CLSM (laser scanning confocal microscope). Referring to the results of SIF incubation for 1-4 hours shown in FIG. 3g, EcN could be detached from the envelope after 4 hours of incubation in SIF in general.
To investigate the in vivo pH response based controlled release behavior of EcN from the envelope in the intestine, mice were orally administered EcN encapsulated with Cy3 labeled L100-55 and capable of expressing GFP. After 1 and 4 hours post-dose, mice were sacrificed and their gastrointestinal tract was restored and examined under an In Vivo Imaging System (IVIS). According to FIG. 3h, the fluorescence signal of Cy3 spread throughout the GI tract 1 hour after oral administration, but the EcN-GFP signal was limited to only a portion of the region L100-55, indicating that the envelope was tightly attached to the EcN surface 1 hour after oral administration. However, the fluorescence signal area of Cy3 became much smaller after 4 hours of oral administration compared to 1 hour, but the position of EcN-GFP extended throughout the entire gastrointestinal tract, and these results indicate that, within 4 hours of administration, the envelope (i.e., the polymer material) dissolved and detached from the surface of EcN in the intestine, and the isolated L100-55 was rapidly excreted from the body, indicating that the envelope can also exhibit strong stability of pH to the intestinal response in vivo.
Example 3
In vitro use of simulated gastric fluid
And intestinal juice
The ability to increase resistance due to the EcN-L envelope in the gastrointestinal tract was tested. The inventors investigated the effect of the assembled layer on the survival and growth of EcN-L in the gastrointestinal environment. As shown in FIG. 4a, EcN-L showed a significant increase in tolerance to both SGFs compared to uncoated ECN, indicating that the coating can protect EcN from the gastric acid environment in the stomach. However, as shown in fig. 4b, the growth rate of EcN-L in SIF clearly lags behind EcN within 3 hours, and the survival rate of both EcN and EcN-L in SIF did not significantly affect as the incubation time was extended to 4 hours, indicating that the intestinal microenvironment was suitable for EcN landing growth, and after 4 hours of incubation in SIF, the coating polymer layer on EcN surface was completely removed.
To investigate whether the pH-responsive polymer envelopes could enhance EcN survival and colonization in vivo, mice were gavaged with EcN with envelopes by oral gavage to study their distribution and retention in the gastrointestinal tract. After 4, 48 and 96 hours, mice were euthanized and EcN counts in the contents and tissues of the stomach, intestine, colon and cecum, respectively, were assessed by plate counting. As shown in FIGS. 4c-4h for bacterial counts and 4i for EcN and EcN-L survival in the gut, EcN-L showed higher survival in the stomach and intestinal tract of mice after 4 hours post-dose compared to EcN without the coating. The retention in the intestinal tract can be maintained for up to 4 days after administration. These data provide strong evidence that the anionic polymer coating of EcN can not only protect EcN from acids and enzymes in the stomach, thereby improving its survival and colonization rates, but also facilitate its targeted intestinal delivery and retention. The gastrointestinal tract of mice after gavage of both uncoated EcN and coated EcN-L was removed and imaged (as shown in FIG. 4 j), the fluorescence signal of the EcN-L gastrointestinal tract region was much stronger than that of the uncoated EcN group, indicating that the pH responsive coating on the surface of EcN was stable in the gastric environment, effectively inhibiting the destruction of ECN in the stomach. EcN-L, after passing through the stomach, the neutral pH of the intestine gradually increases, greatly increasing its oral bioavailability by deprotonating the functional groups of the polymer and increasing its solubility, resulting in dissolution of the protective coating, which then detaches from the EcN surface, resulting in targeted delivery of the controlled release EcN to the intestine.
Furthermore, we also investigated the retention of EcN with thicker envelopes (anionic polymer) and TEM results showed that, as shown in figure 5a, when EcN-L was incubated in SIF, it took 10 hours to completely remove the envelope, which was much longer than the thin envelope (4 h); additionally, EcN amounts were also calculated in stomach, intestine, colon and cecum contents and tissues, and the results indicated that EcN-L with thick membranes was much less viable than uncoated EcN (FIGS. 5b-5e), since the coating on EcN was difficult to remove due to poor response to pH to prevent bacteria from directly contacting the intestinal mucosa, thereby affecting EcN's ability to colonize in the intestine. That is, the pH responsiveness of the coating plays a key role in improving the retention efficiency of EcN.
Example 4
In the clinical setting, bacterial infections are a major cause of morbidity and mortality. The widespread emergence of drug resistant strains exacerbates their effects. If administered orally, antibiotics are easily used to treat pathogen infections, which makes it difficult to maintain high concentrations at the site of intestinal infection, and also eliminates beneficial microorganisms and results in the persistence of resistant strains and disrupts the balance of the intestinal flora, especially broad spectrum antibiotics. As reported, escherichia coli Nissle 1917 is a microorganism that confers beneficial physiological and therapeutic activities. Importantly, the probiotic bacteria will target the micro-organisms or their toxins with high specificity to kill pathogens and modulate the intestinal flora balance. Based on controlled release and targeted delivery of EcN (EcN-L) with an envelope, the inventors focused on the ability of enveloped EcN to overcome intestinal infections, and evaluated the effect of enveloped EcN on colonization of the intestinal pathogen, e.coli K88, infection in vitro and in vivo.
As shown in fig. 6a-c, the survival of K88 when EcN and EcN-L were incubated with K88 demonstrated that the reduced growth rate was due to the result of effective killing of pathogens in the system, indicating that EcN had a higher inhibitory activity against K88. Next, the inventors investigated the therapeutic effect by administering EcN or enveloped EcN to mice previously infected with K88. A count of K88 was obtained as shown in figure 6d, and a sharp decrease in K88 levels in feces over time was observed after feeding EcN-L and EcN mice compared to the infected control group, noting that K88 was decreased more rapidly in the EcN-L group than in the EcN group. After sacrifice, intestinal contents and tissues were collected for examination (fig. 6e-f), and at the same time oral EcN and EcN-L treatments resulted in a greater decline in K88 counts in the gastrointestinal tract than in the infected group alone. The reduction in K88 in group EcN-L was statistically significant compared to group EcN. Fig. 6g and 6h also conclude that enveloped EcN produced a more pronounced colonization defect for K88 in intestinal epithelial cells. All these results indicate that coated EcN has potent pathogen-specific killing activity in intestinal infections, suggesting that controlled release and targeted delivery of coated EcN makes it possible to play an important role in increasing EcN intestinal colonization, which is also the determining factor to reduce pathogen burden in the intestine.
Claims (9)
1. A method for producing a surface-modified bacterium having a surface coated with a coating film, comprising the steps of,
treating the bacteria with a salt solution to allow the bacteria surface to adsorb cations,
adding a polymer which can act with cation static electricity to form a coating on the surface of the bacteria by self-assembly.
2. The method of producing a surface-modified bacterium according to claim 1, comprising the steps of,
treatment of bacteria with calcium salt solutions to adsorb Ca on the surface of the bacteria2+The ions are selected from the group consisting of,
adding a polymer to self-assemble on the surface of the bacteria to form a coating;
preferably, the bacteria are treated with a calcium phosphate solution comprising calcium chloride to allow Ca to be adsorbed on the surface of the bacteria2+The ions are selected from the group consisting of,
the anionic polymer solution is added and then the pH is lowered to enable complete self-assembly on the bacterial surface to form a coating.
3. The method of claim 2, wherein the pH drop is 5 or less.
4. The method of claims 1-3, wherein the anionic polymer is preferably a pH-responsive anionic polymer, preferably an anionic polymer L100-55.
5. A surface-modified bacterium having a surface coating thereon, wherein the surface coating comprises
A polymer; and the combination of (a) and (b),
calcium ions for crosslinking the anionic polymer.
6. The surface-modified bacterium according to claim 4, wherein the polymer is a pH-responsive polymer, an enzyme-responsive polymer, a light-responsive polymer, a reductively-responsive polymer; preferably, the polymer is a pH-responsive anionic polymer, preferably, the pH-responsive anionic polymer is capable of dissolving at a pH >5.5, preferably, the pH-responsive anionic polymer is capable of dissolving at a pH > 6; after being wrapped, the bacteria are temporarily inactivated when the pH is less than 5.5, the environment can be intelligently identified, and when the pH is more than 5.5, the bacteria can be quickly revived after the outer membrane is removed in a response manner, so that the bacteria can be controlled to be released and targeted delivery can be realized;
preferably, the pH-responsive anionic polymer is an anionic polymer L100-55.
7. A method for improving the intestinal flora structure by oral or gastrointestinal administration of the surface-modified bacteria of claim 5 or 6, which is resistant to attack by gastric acid and digestive enzymes, to inhibit the growth and/or colonization of intestinal pathogens and to selectively increase the content and oral availability of certain bacteria in the intestine; preferably, the bacteria are selected from the group consisting of probiotics, preferably one or more combinations of escherichia coli, enterobacter faecalis, staphylococcus aureus, probiotic bacillus, clostridium butyricum, lactobacillus, bifidobacterium and actinomycetes.
8. The method for improving gut flora structure of claim 7, further comprising orally or enterally administering an antibacterial agent.
9. Method for treating intestinal inflammation and/or improving intestinal function, characterized in that surface-modified bacteria according to claim 5 or 6 are administered orally or gastrointestinal to inhibit the growth of intestinal pathogens and/or to eliminate pathogens.
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| CN115737563A (en) * | 2022-11-21 | 2023-03-07 | 清华大学深圳国际研究生院 | Preparation method of liposome-coated surface-modified microorganism |
| CN116926054A (en) * | 2023-06-06 | 2023-10-24 | 广东亿超生物科技有限公司 | Armoured probiotics with catalytic function and preparation method thereof |
| CN116926054B (en) * | 2023-06-06 | 2024-01-23 | 广东亿超生物科技有限公司 | Armoured probiotics with catalytic function and preparation method thereof |
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