CN111996148B - 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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- CN111996148B CN111996148B CN202010910772.4A CN202010910772A CN111996148B CN 111996148 B CN111996148 B CN 111996148B CN 202010910772 A CN202010910772 A CN 202010910772A CN 111996148 B CN111996148 B CN 111996148B
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- A61K35/74—Bacteria
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- A61K35/742—Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
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- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
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- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
- A61K35/745—Bifidobacteria
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- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
- A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
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- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/005—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor after treatment of microbial biomass not covered by C12N1/02 - C12N1/08
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The present invention relates to a method for preparing a surface-modified microorganism, the surface of which has an envelope, comprising the steps of treating the bacteria with a salt solution to cause the surface of the bacteria to adsorb cations; the anionic polymer is added to form an envelope on the surface of the bacteria by self-assembly, so that the bacteria are protected from being corroded by gastric acid, bile acid and digestive enzymes, and the oral availability of the bacteria is greatly improved; meanwhile, the bacteria after being wrapped are temporarily inactivated, and the surrounding environment responsiveness can be intelligently identified to remove the capsule, 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, relates to bacteria with surface modification, a preparation method and application thereof, and in particular relates to bacteria with surface coating, a preparation method and application thereof.
Background
Naturally, various microflora colonize the human body, and these microorganisms inhabit our body and build a complex ecosystem to affect 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 considered to be affected by the composition of the microbiota. Because bacteria can interact with their niches in the human body and can also produce some key biomolecules, they react 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 very important microorganism management tools for the host as viable microorganisms, are useful in the treatment and diagnosis of diseases to regulate the immune system, cellular metabolism, and epithelial barrier function, etc. However, not all probiotics are beneficial in all cases, and we still face many challenges for clinical transformations when developing microbiota targeted therapies, developing techniques to enhance efficacy, and providing methods of targeted delivery to specific tissues to ensure biocompatibility and therapeutic efficacy are critical.
The intestinal tract is the largest pool of microorganisms in the human body. Intestinal flora affects many aspects of host physiology and is believed to be a key factor in mediating host adaptive 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, delivery of probiotics to the gut to affect and regulate gut microbial composition can potentially affect the treatment of many human diseases. Oral probiotics are considered convenient and widely accepted and patient compliance is enabled. However, for oral probiotics, the challenge encountered with 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 probiotics. A fatal disadvantage of current commercial probiotic products is that insufficient delivery of probiotics to the lower gastrointestinal tract, especially the acidic environment of the stomach, limits the conversion of many probiotic delivery techniques during oral delivery. Because of the limited efficacy of probiotics currently used by oral delivery, the ability of probiotics to protect them from acids and enzymes in the stomach and target delivery to the gut for a clear, health-promoting purpose would be of great value. Microencapsulation has heretofore been defined as the primary technique to address the challenges of oral administration of probiotics to prevent direct contact of the probiotics with the harsh environment of the gastric tract. Unfortunately, this approach also prevents direct interaction of probiotics with the intestinal surface, thereby reducing their colonization in the intestinal tract. In particular, the over-introduced microencapsulated drugs can also have a series of adverse effects on the biological activity of the probiotics and host health. Thus, this approach would not have sufficient advantage in animal models or human transformation.
Disclosure of Invention
In view of the above-described problems of the prior art, the inventors have provided surface-modified bacteria and methods for their preparation, in particular bacteria having a surface film (composed of a polymer), which ensure that bacteria (e.g. probiotics) are temporarily inactivated in the stomach, so that they can overcome the acid and enzyme injuries present in the stomach; when delivered to the intestine, the encapsulated bacteria automatically remove their envelopes in the intestine and resume their activities as desired, with the aim of selectively releasing the bacteria, in particular probiotics, to the targeted lower digestive tract, depending on the low pH and digestive enzymes in the digestive tract that cause denaturation.
In one aspect the invention relates to a method for the preparation of a surface modified bacterium having an envelope on the surface of said bacterium, comprising the steps of,
the bacteria are treated with a salt solution to allow the bacteria surface to adsorb cations,
polymers are added which are capable of electrostatically interacting with cations to enable self-assembly at the bacterial surface to form an envelope.
Preferably, the method for preparing the surface modified bacteria according to the present invention comprises the steps of:
treatment of bacteria with calcium salt solution to allow adsorption of Ca on the bacterial surface 2+ Ions;
polymers are added to self-assemble at the bacterial surface to form the envelope.
Preferably, the bacteria are treated in suspension with a calcium phosphate solution containing calcium chloride so that the surfaces of the bacteria adsorb Ca 2+ Ions;
an anionic polymer solution is added and then the pH is lowered to enable self-assembly to form an envelope on the bacterial surface.
Preferably, the pH is reduced to 5 or less; further preferably, the anionic polymer is preferably anionic polymer L100-55.
In another aspect, the invention relates to a surface-modified bacterium having an envelope on its surface, wherein said envelope comprises:
a polymer; and, a step of, in the first embodiment,
calcium ions for crosslinking the anionic polymer.
Preferably, the polymer is a pH responsive polymer, an enzyme responsive polymer, a light responsive polymer, a reducing responsive polymer; preferably, the polymer is a pH-responsive anionic polymer, preferably the pH-responsive anionic polymer is capable of dissolution at a pH >5.5, preferably the pH-responsive anionic polymer is capable of dissolution at a pH > 6. Bacteria are temporarily inactivated when the pH is less than 5.5 after encapsulation, the environment can be intelligently identified, and the bacteria can be quickly revived after the outer membrane is removed in a responsive manner when the pH is more than 5.5, so that the bacteria can be controlled to be released and targeted delivery can be realized;
preferably, the pH responsive anionic polymer is anionic polymer L100-55.
The present invention also relates to a method for improving the structure of intestinal flora, which is capable of withstanding gastric acid and attack by digestive enzymes by oral or gastrointestinal administration of the above surface-modified bacteria, to inhibit the growth and/or colonization of intestinal pathogens and to 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 EcN, escherichia coli, probiotics, clostridium butyricum, lactobacillus, bifidobacterium and actinomycetes.
Preferably, the method for improving the structure of intestinal flora further comprises orally or enterally administering an antibacterial agent.
The invention also relates to a method for treating intestinal inflammation and/or improving intestinal function, characterized in that the above surface-modified bacteria are administered orally or parenterally to inhibit the growth of intestinal pathogens and/or to eliminate pathogens.
Drawings
FIG. 1a is a schematic diagram of a method of producing an enveloped bacterium according to the present invention;
FIGS. 1b-c are transmission electron microscope images of enveloped bacteria in accordance with the present invention;
fig. 1d is a transmission electron microscope image of bacteria with envelopes formed in PBS at ph=7.4 according to the present invention;
FIG. 1e is an image of a laser scanning confocal microscope of bacteria having an envelope in an embodiment of the invention;
FIG. 1f is a graph showing the growth of bacteria having an envelope in an embodiment of the present invention;
FIG. 1g shows the zeta potential change of bacteria with an envelope according to an embodiment of the invention;
FIG. 2 is a transmission electron microscope image of bacteria having different bacterial thicknesses in an embodiment of the present invention;
FIGS. 3a-b are schematic illustrations of the temporary inactivation of encapsulated bacteria at lower pH in embodiments of the present invention;
FIGS. 3c-d are schematic illustrations of on-demand reactivation of encapsulated bacteria at higher pH in an embodiment of the invention;
FIGS. 3e and 3f illustrate the removal of the coating from the bacterial surface in an embodiment of the present invention;
FIG. 3g is an image of a confocal laser scanning microscope of SIF in which the incubation coating is detached from the bacterial surface in an embodiment of the invention;
FIG. 3h shows the change in the gastrointestinal tract of bacteria having an envelope in accordance with an embodiment of the present invention;
FIG. 4a shows tolerance to simulated gastric fluid by bacteria having an envelope in an embodiment of the invention;
FIG. 4b shows growth of EcN and enveloped bacteria (EcN-L) in intestinal fluid (SIF) in accordance with embodiments of the present invention;
FIGS. 4c-4h show the survival numbers of enveloped bacteria (EcN-L) and non-enveloped bacteria EcN in the stomach and intestinal tract of mice in accordance with embodiments of the present invention;
FIG. 4i shows the survival rates of enveloped (EcN-L) and non-enveloped EcN bacteria in the stomach and intestinal tract of mice in an embodiment of the present invention;
FIG. 4j is an in vitro imaging of enveloped bacteria (EcN-L) and non-enveloped bacteria from the gastrointestinal tract in accordance with an embodiment of the present invention;
FIG. 5a shows retention of EcN with thicker envelopes in an embodiment of the invention;
FIGS. 5b-5e are viability assays of EcN-L with thicker coating and EcN without coating in embodiments of the present invention;
FIGS. 6a-6c are graphs showing the survival of K88 when EcN and enveloped bacteria EcN-L are incubated with K88 in an embodiment of the invention;
FIG. 6d shows the amount of K88 in the faeces of mice fed EcN-L and EcN according to an embodiment of the invention;
FIGS. 6e-6f are counts of K88 after collection of intestinal content and tissue following mice sacrificed in an embodiment of the present invention;
FIGS. 6g and 6h show the effect of bacteria with an envelope on K88 colonization in the gut in an embodiment of the invention.
Detailed Description
The present application is described in further detail below with reference to the drawings and embodiments. It is to be understood that the specific embodiments described herein are merely illustrative of the technical solution, inventive concepts, and not restrictive of the invention. In addition, for convenience of description, only a portion related to the invention is shown in the drawings. Other parts not explicitly shown or explicitly described are understood to be prior art conventional means or arrangements which, in combination with the technical features shown in the present invention, may achieve the technical effects of the present invention.
It should be noted that, without 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 accompanying drawings in conjunction with embodiments.
The surface-modified bacteria of the invention may be coated with exogenous, natural, non-natural films. The surface-modified bacteria according to the embodiments of the present invention have an envelope on the surface thereof, and the envelope is formed of a polymer. In a preferred embodiment of the present application, the surface-modified bacteria of the present invention have a surface coating formed of an anionic polymer (preferably L100-55), and the surface of the bacteria is first coated with cations during the preparation process, and then the anionic polymer electrostatically reacts with the cations on the surface of the bacteria to self-assemble the coating, for example, in a specific embodiment of the present application, the bacteria are treated with a calcium salt solution (e.g., a calcium chloride solution) to cause Ca adsorption on the surface of the bacteria 2+ Ions, followed by addition of anionic polymers to self-assemble at the bacterial surface to form the envelope. As shown in the schematic flow chart of FIG. 1a, ca is adsorbed on the bacterial surface 2+ After the addition of the ion, the pH is lowered to enable the anionic polymer to react with Ca 2+ Electrostatic interaction, self-assembly on the bacterial surface to form the envelope. In particular embodiments of the invention, the bacteria are treated (e.g., suspended) using a calcium phosphate solution comprising calcium chloride. The inventors have found that when the pH reduction is less than or equal to 5, the integrity of the coating is superior (relative to environments with pH above 5, for example ph=7.4 in the specific example).
The bacteria may be probiotics or medical strains. In this embodiment, the bacteria may be one or more of escherichia coli EcN, enterococcus faecalis, bacillus subtilis, clostridium butyricum, lactobacillus, bifidobacterium and actinomycetes; for example E.coli Nissle 1917 (E.coli Nissle 1917, ecN), B.subtilis, E.faecalis (Enterococcus faecalis).
The bacterial surface has an envelope, formed with particular reference to the preparation methods described above or in particular examples, the envelope formed 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 envelope according to the present invention can dissolve, disintegrate or disintegrate at pH >5.5 or pH >6, for example at ph=6.8, and can control release of bacteria and targeted delivery of bacteria, for example by protecting bacteria from disintegration and survival when passing gastric acid, and can disintegrate and disintegrate in the intestinal tract, thereby releasing bacteria. In other embodiments of the invention, the polymer forming the coating may also be an enzyme-responsive polymer, a light-responsive polymer, a reductive-responsive polymer. The enzyme-responsive polymer is degradable in the presence of certain enzymes (e.g., certain digestive enzymes, hydrolytic enzymes, etc.), the light-responsive polymer is degradable or degradable under light conditions, and the reducing-responsive polymer is degradable or degradable in the presence of a reducing enzyme or reducing agent, thereby being degradable at a specific location (in the gut). The surface-modified bacteria disclosed by the invention are coated on the surface, the bacteria are temporarily inactivated when the pH is less than 5.5, the environment can be identified, and the bacteria can be rapidly compounded after the outer membrane is removed in a responsive manner when the pH is more than 5.5, so that the bacteria can be released in a controlled manner and targeted delivery can be realized.
According to the characteristics of the bacteria having an envelope of the present invention, the bacteria can be used to improve the intestinal flora structure, for example, by orally or parenterally administering the surface-modified bacteria (i.e., bacteria having an envelope) which are resistant to attack by gastric acid and digestive enzymes and which are released and proliferate in the intestinal tract to inhibit the growth and colonization of pathogenic bacteria in the intestinal tract, reduce the number of pathogenic bacteria, and increase the content and oral availability of beneficial bacteria (probiotics) in the intestinal tract, and finally improve the intestinal flora structure. Among them, bacteria may be selectively increased, and escherichia coli EcN, escherichia coli, probiotics, clostridium butyricum, lactobacillus, bifidobacterium and actinomycetes may be selected or used in combination. According to the conventional practice of current medical treatment, the above surface-modified bacteria may be administered after oral or enteral administration of antibacterial agents. The antibacterial agent may be a natural drug or an extract thereof, for example, derived from berberis, coptis, etc., or an antibiotic, for example, quinolones, beta-lactams, macrolides, etc. In addition, the bacteria may be used to treat intestinal inflammation and/or to improve intestinal function, i.e., orally or parenterally administered to inhibit the growth of intestinal pathogens and/or to eliminate pathogens, which may be used in combination with antibacterial agents.
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 E.coli EcN), and E.coli K88 (hereinafter K88) were purchased from China general microbiological culture collection center (GMCC). Plasmids pBBR1MCS2-Tac-mCherry (kanamycin resistance) and pMP-2444-GFP (gentamicin resistance, expressing GFP). Commercial Luria-Bertani (LB) as cloning medium, 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 bacterial culture was washed and resuspended in 1ml containing 12.5mM CaCl 2 To ice-cold calcium phosphate solution, then the appropriate L100-55 solution is added. After mixing for several minutes, the pH of the resulting solution was reduced toColi EcN with an envelope (coating), i.e. bacteria with an envelope, was obtained by centrifugation re-suspension (using dilute hydrochloric acid) (called EcN-L) and washed in PBS (pH 5). As shown in FIG. 1a, ca 2+ The ions adsorb to the surface of EcN due to the negative charge on its surface, thereby converting the surface of EcNIs positively charged to form cross-linking sites. Then, according to the anionic polymer L100-55 and Ca 2+ Electrostatic interactions between them when PBS pH<When 5.5 is lowered, the anionic polymer L100-55 will self-assemble at the surface of 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 sample was then completely dried in air prior to observation. Then, ecN-L and EcN were further visualized by a laser scanning confocal microscope (Leica TCS SP8, germany) with Cy 3-labeled L100-55 and Green Fluorescent Protein (GFP) expressed in EcN. The average size and apparent Zeta potential of EcN-L and EcN were determined by dynamic light scattering (Malvern Zetasizer nano ZS, uk).
As shown in fig. 1b and 1c, the transmission electron microscope image shows EcN with a thin and transparent outer layer (envelope) covering the surface, indicating that a small amount of polymer can achieve complete coverage for bacteria. As shown in FIG. 2, by varying the amount of L100-55 (20. Mu. L L100 in FIG. 2a and 30. Mu. L L100 in FIG. 2b in 100-55), the thickness of the coating on the bacterial surface will also vary, i.e., the thickness of the coating assembled onto the bacterial surface can be effectively adjusted.
The integrity of the coating depends on the pH at which it is located, for example the pH of the PBS solution used for storage and re-suspension, the bacterial coating formed in PBS at ph=7.4 shown in fig. 1d has less integrity than the bacterial coating formed at ph=5 (shown in fig. 1 b). EcN-L was further visualized by Laser Scanning Confocal Microscopy (LSCM) with the dye Cy3 labeling L100-55. As shown in FIG. 1e, GFP (green) expressing EcN was quantitatively enveloped by L100-55 (red). Dynamic Light Scanning (DLS) measurements showed an increase in size after EcN encapsulation of L100-55, but a decrease in zeta potential from-15.6 mV to-17 mV (FIG. 1 g), indicating the presence of anionic carboxyl groups of L100-55 on the EcN surface. To check if the wrapped envelope (polymer layer) has an effect on the viability of EcN, a growth curve is recorded (fig. 1 f). The growth rate of coated EcN was slightly retarded within the first 4 hours compared to uncoated EcN due to the fact that the growth was limited by the incompletely removed coating on the probiotic surface. Subsequently, ecN-L reached the same OD600 growth rate at 5h as uncoated EcN, indicating that the coating was completely removed and had a negligible effect on bacterial viability after coating EcN with L100-55.
Example 2
Coated EcN was tested for on-demand reactivation, controlled release and pH response stability. As shown in fig. 3a-b, when the encapsulated bacteria were cultured at 37 ℃ in LB at ph=5, the bacteria were difficult to grow, exhibiting temporary inactivation; and when it was incubated in LB with ph=7 (fig. 3 c-d) at 37 ℃, the bacteria grew rapidly after demoulding, exhibiting the property of reviving as required. As shown in the TEM image (fig. 3 e), when the coated EcN was incubated in SIF at ph=6.8, the behavior of the coating from swelling to rupture and finally complete removal from the EcN surface was observed, which process took about 4 hours. The length of time indicated that coated ECN showed great potential in controlling EcN release into the targeted intestine, and that bacterial morphology remained intact after SIF exposure. The responsive release of the encapsulated EcN in SIF was also monitored by flow cytometry (fig. 3 f).
The pH response stability of the envelope (anionic polymer) of the envelope-coated EcN in SIF was examined using CLSM (laser scanning confocal microscope). Referring to the results of 1-4 hours incubation of SIF shown in fig. 3g, generally EcN can be released from the envelope after 4 hours incubation in SIF.
To investigate the controlled release behavior of coated EcN in the intestine based on in vivo pH response, mice were orally coated with Cy 3-labeled L100-55 and capable of expressing GFP EcN. After 1 and 4 hours post-dose, mice were sacrificed and their gastrointestinal tract restored for examination under an In Vivo Imaging System (IVIS). According to FIG. 3h, the fluorescence signal of Cy3 was distributed throughout the gastrointestinal tract after 1 hour of oral administration, but the EcN-GFP signal was limited to only a portion of the L100-55 region, indicating that the envelope was tightly attached to the EcN surface 1 hour after oral administration. However, after 4 hours of oral administration, the fluorescence signal region of Cy3 became much smaller compared to 1 hour, but the position of EcN-GFP was spread throughout the stomach, and these results indicate that, within 4 hours of administration, the envelope (i.e., the polymer material) was dissolved and detached from the surface of the intestine EcN, and the separated L100-55 was then rapidly discharged from the body, indicating that the envelope was also able to exhibit a strong stability of pH response to the intestinal tract in the body.
Example 3
In vitro use of simulated gastric fluidHe Zhi Ye (gastrointestinal fluid)>To test the ability to enhance resistance due to the EcN-L envelope in the gastrointestinal tract. 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 non-coated ECNs, indicating that the coating can protect EcN from gastric acid environment in the stomach. However, as shown in FIG. 4b, the growth rate of EcN-L in SIF significantly lags EcN in 3 hours, and the survival rate of EcN and EcN-L in SIF has no significant effect as the incubation time is extended to 4 hours, indicating that the intestinal microenvironment is suitable for the landing growth of EcN, and the coated polymer layer on the EcN surface can be completely removed after incubation in SIF for 4 hours.
To investigate whether the pH-responsive polymer coating could enhance survival and colonisation in vivo EcN, mice were gavaged with coated EcN by oral gavage to investigate their distribution and retention in the gastrointestinal tract. After 4, 48 and 96 hours, mice were euthanized and the number of EcN in the contents and tissues of the stomach, intestine, colon and cecum, respectively, were assessed by plate counting. The numbers of bacteria shown in FIGS. 4c-4h and the survival rates of EcN and EcN-L in the digestive tract shown in FIG. 4i showed a higher survival rate of EcN-L in the stomach and intestinal tract of mice 4 hours after administration compared to EcN without the coating. Up to 4 days of retention in the intestine can be maintained 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 colonisation rate, but can also promote its targeted intestinal delivery and retention. The gastrointestinal tract of mice after lavage of uncoated EcN and coated EcN-L was removed and imaged (as shown in FIG. 4 j), the fluorescence signal of the gastrointestinal tract region of EcN-L 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 destruction of ECN in the stomach. EcN-L, after passing through the stomach, gradually increases the neutral pH of the intestine, leading to dissolution of the protective coating by deprotonating the functional groups of the polymer and increasing its solubility, and then release from the EcN surface, leading to targeted delivery of the controlled release EcN to the intestine, greatly increasing its oral bioavailability.
Furthermore, we also studied the retention of EcN with thicker coating (anionic polymer), and the TEM results show that it takes 10 hours to completely remove the coating when EcN-L is incubated in SIF, as shown in fig. 5a, which is much longer (4 h) than the thin coating; in addition, the amounts of EcN in the stomach, intestine, colon and cecum contents and tissues were also calculated, and the results showed that EcN-L with thick film had much lower viability than uncoated EcN (fig. 5b-5 e) because the coating on EcN was difficult to remove due to poor pH responsiveness preventing bacteria from directly contacting the intestinal mucosa, thereby affecting the ability of EcN to colonise the intestine. That is, the pH responsiveness of the coating plays a key role in improving the retention efficiency of EcN.
Example 4
Bacterial infections are a major cause of morbidity and mortality in the clinical setting. The widespread emergence of drug resistant strains exacerbates their effects. If the antibiotic is administered orally, it is easy to use the antibiotic for treating pathogen infection, which makes it difficult to maintain high concentration at the site of intestinal infection, and oral antibiotics can also eliminate beneficial microorganisms and cause the persistence of drug-resistant strains, and disrupt the balance of intestinal flora, especially broad-spectrum antibiotics. As reported, escherichia coli Nissle 1917 is a microorganism that confers beneficial physiological and therapeutic activity. Importantly, the probiotics will target the microorganism or its toxins with high specificity to kill pathogens and regulate intestinal flora balance. Based on the controlled release and targeted delivery of EcN (EcN-L) with an envelope, the inventors focused on the ability of the envelope EcN to overcome intestinal infections, evaluating the effect of the envelope EcN on colonization of intestinal pathogen infections in vitro and in vivo.
As shown in fig. 6a-c, when EcN and EcN-L were incubated with K88, the survival of K88 demonstrated that the reduction in growth rate was due to the effect of efficient killing of pathogens in the system, indicating that EcN had higher inhibitory activity on K88. Next, the inventors studied the effect of treatment by administering EcN or enveloped EcN to mice previously infected with K88. As shown in fig. 6d, a sharp drop in K88 levels in feces over time was observed after feeding the EcN-L and EcN mice compared to the infected control group, and it should be noted that K88 fed EcN-L dropped faster than EcN. After mice were sacrificed, intestinal contents and tissues were collected for examination (fig. 6 e-f), and at the same time oral EcN and EcN-L treatments resulted in a decrease in K88 counts in the gastrointestinal tract that was greater than in the infected group alone. The K88 reduction in EcN-L groups was statistically significant compared to EcN groups. FIGS. 6g and 6h also show that coated EcN produced a more pronounced defect in colonisation of 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 enables it to play an important role in increasing intestinal colonisation of EcN, which is also a determinant in reducing pathogen burden in the gut.
Claims (6)
1. A method for preparing a surface-modified bacterium having an envelope on the surface thereof, comprising the steps of treating the bacterium with a salt solution to cause the bacterium surface to adsorb cations, adding a polymer capable of electrostatically reacting with the cations to be capable of self-assembling on the bacterium surface to form an envelope;
comprising the steps of treating bacteria with a calcium salt solution to cause the surfaces of the bacteria to adsorb Ca 2+ Ions, adding polymers to self-assemble on the bacterial surface to form an envelope; treating bacteria with a calcium phosphate solution containing calcium chloride to cause the surfaces of the bacteria to adsorb Ca 2+ Ions, adding an anionic polymer solution, and then reducing the pH value to be capable of completing self-assembly on the surface of bacteria to form an envelope;
the pH is reduced to 5 or less;
the anionic polymer is pH responsive anionic polymer, and the anionic polymer L100-55.
2. The surface-modified bacterium produced by the method of claim 1, having an envelope on the surface thereof, wherein said envelope comprises a polymer; and calcium ions for crosslinking the anionic polymer.
3. The surface modified bacterium of claim 2, wherein said polymer is a pH-responsive polymer, an enzyme-responsive polymer, a light-responsive polymer, a reductive-responsive polymer; the polymer is a pH responsive anionic polymer, the pH responsive anionic polymer is capable of dissolving at a pH >5.5, and the pH responsive anionic polymer is capable of dissolving at a pH > 6; bacteria are temporarily inactivated when the pH is less than 5.5 after encapsulation, the environment can be intelligently identified, and the bacteria can be quickly revived after the outer membrane is removed in a responsive manner when the pH is more than 5.5, so that the bacteria can be controlled to be released and targeted delivery can be realized;
the pH responsive anionic polymer is anionic polymer L100-55.
4. Use of a surface modified bacterium according to claim 2 or 3 for the manufacture of a medicament for improving the structure of the intestinal flora, which medicament is capable of tolerating gastric acid and digestive enzymes for oral or gastrointestinal administration, to inhibit the growth and/or colonisation of intestinal pathogenic bacteria and to selectively increase the content and oral availability of a certain bacterium in the intestinal tract; the bacteria are selected from probiotics, and the probiotics are one or more of escherichia coli EcN, escherichia coli, probiotics spore bacteria, clostridium butyricum, lactobacillus, bifidobacterium and actinomycetes.
5. Use of the surface modified bacterium of claim 4 in the manufacture of a medicament for improving the structure of the intestinal flora, further comprising oral or enteral administration of an antibacterial agent.
6. Use of a surface modified bacterium according to claim 2 or 3 for the manufacture of a medicament for improving the structure of the intestinal flora for the treatment of intestinal inflammation and/or for improving intestinal function, wherein the surface modified bacterium according to claim 2 or 3 is administered orally or parenterally for inhibiting the growth of and/or for eliminating pathogenic bacteria.
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