Brain-targeting drug delivery system based on outer membrane of engineered escherichia coli dh5a and preparation method and application thereof

Abstract

The application discloses a brain-targeting drug delivery system based on a modified outer membrane of Escherichia coli DH5a and a preparation method and application thereof. The drug delivery system is based on specific recognition and combination between outer membrane protein A of the Escherichia coli DH5a and gp96 on endothelial cells of a blood-brain barrier, and mediates the drug delivery system to penetrate the blood-brain barrier and finally distribute in intracranial interstitial tissue. The brain-targeting drug delivery system can penetrate the blood-brain barrier to reach the brain parenchyma through a transcellular vesicular transport pathway, the preparation method is simple, the drug delivery system has good biological safety and superior delivery efficiency, and the drug delivery system has high operability and considerable economic benefits.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of nanobiomedicine, and particularly relates to a brain-targeting drug delivery system based on a modified outer membrane of Escherichia coli DH5a and a preparation method and application thereof. BACKGROUND

[0002] The blood-brain barrier (BBB) is a barrier between plasma and brain cells composed of tightly connected brain microvascular endothelial cells, pericytes, astrocytes and basement membranes, which is mainly surrounded by extracellular matrix composed of collagen and laminin. There are tight junctions between vascular endothelial cells, which limit the entry of many substances from the intercellular space (paracellular pathway) into the brain. These components help maintain the special properties of the blood-brain barrier, providing various growth and differentiation factors required by endothelial cells. The main function of the blood-brain barrier is to selectively permeate substances, prevent potentially harmful substances from entering the brain, and allow nutrients and signaling factors to pass normally, thereby maintaining a stable environment in the brain. If the integrity of the blood-brain barrier cannot be maintained, it will cause abnormal reactions in the body and even serious pathological phenomena, such as meningitis, multiple sclerosis, neurodegenerative diseases, etc. The blood-brain barrier blocks blood-borne neurotoxins and exogenous toxic substances to protect the central nervous system (CNS), but also limits the delivery of drugs to the CNS to exert therapeutic effects, adding difficulty in treatment. So far, there have been many reports on the delivery of new nanomaterials targeting the brain. However, the accumulation of most engineered nanoparticles in the brain is still less than 1%, which indicates that the delivery system still needs to be further improved. A large number of literatures show that Gram-negative bacteria Escherichia coli K1 can cross the BBB and colonize the brain, thereby inducing bacterial meningitis. Therefore, it is the research direction to extract the outer membrane protein of the bacteria that can penetrate the blood-brain barrier by the invasion of meningitis bacteria to establish an efficient nanodrug delivery system. In this research direction, how to safely use the outer membrane vesicles of the bacteria that cause meningitis and transformation is a problem to be solved. SUMMARY

[0003] The application aims to provide a brain-targeting drug delivery system based on a modified outer membrane of Escherichia coli DH5a and a preparation method and application thereof.

[0004] The technical scheme of the application is as follows:

[0005] The brain-targeting drug delivery system based on a modified outer membrane of Escherichia coli DH5a uses biodegradable high molecular materials to prepare drug-loaded nanoparticles as a basic carrier, and the surface of the basic carrier is wrapped with a modified outer membrane of Escherichia coli DH5a, and the biodegradable high molecular material is polylactic acid-glycolic acid.

[0006] Another technical solution of the present application is:

[0007] The preparation method of the brain-targeting drug delivery system based on the modified outer membrane of E.coli DH5a comprises the following steps:

[0008] (1) Extraction of the modified outer membrane of E.coli DH5a: E.coli DH5a is cultured overnight in a culture medium to make the OD600 value reach 1.5, and the E.coli DH5a bacterial body precipitate is collected by centrifugation; the bacterial body precipitate is resuspended in a phosphate buffer, and is broken by ultrasonic to obtain a broken bacterial liquid; the broken bacterial liquid is centrifuged to obtain a precipitate; Tris buffer is added to the precipitate, and the supernatant is collected by high-speed centrifugation; the supernatant is centrifuged again at high speed to obtain the modified outer membrane of E.coli DH5a with removed lipopolysaccharide; the modified outer membrane of E.coli DH5a is dispersed in ultrapure water for storage;

[0009] (2) Preparation of the nano-carrier: a high-molecular material is weighed and dissolved in an organic solvent to form an oil phase; a drug is dissolved in the oil phase to become part of the oil phase, and the water phase is water itself, or the drug is dissolved in water to form a water phase; the water phase is added dropwise into the vortexed oil phase, and is emulsified by ultrasonic to form a water-in-oil emulsion; the emulsion is added dropwise into the vortexed outer water phase, and is emulsified by ultrasonic to form a water-in-oil-in-water double emulsion; the double emulsion is quickly poured into the volatile water phase, and is stirred overnight to volatilize to form a nano-carrier suspension; the nano-carrier suspension is purified by high-speed centrifugation to obtain a nano-carrier precipitate; the nano-carrier precipitate is dispersed in water by ultrasonic, and is centrifuged at high speed to obtain the final precipitated nano-carrier; the final precipitated nano-carrier is dispersed in ultrapure water;

[0010] (3) Preparation of the nano-carrier wrapped by the outer membrane of E.coli DH5a: the modified outer membrane of E.coli DH5a is mixed with the final precipitated nano-carrier after ultrasonic and is emulsified by ultrasonic, and is extruded multiple times by using a polycarbonate membrane to obtain the brain-targeting drug delivery system based on the modified outer membrane of E.coli DH5a.

[0011] Further, in step (1), the culture medium is a pH 7.4 LB culture medium, and the volume ratio of the bacterial body precipitate to the phosphate buffer is 1:4, and the pH value of the phosphate buffer is 7.4.

[0012] Further, in step (1), the centrifugation temperature of the centrifugal collection of E. coli DH5α bacterial body precipitate is 4℃, the centrifugal force is 5000g, and the time is 10min; the ultrasonic crushing power is 300w, and the time is 30min; the centrifugation temperature of the crushed bacterial liquid is 4℃, the centrifugal force is 2900g, and the time is 1h; the Tris buffer solution is added to the precipitate, the centrifugation temperature of high-speed centrifugation is 4℃, the centrifugal force is 20000g, and the time is 1h; the centrifugation temperature of the supernatant high-speed centrifugation again is 4℃, the centrifugal force is 125000-150000g, and the time is 2h.

[0013] Further, in step (1), the Tris buffer solution added to the precipitate is specifically: 7.5 times the volume of 0.1M Tris buffer solution based on the wet weight of the precipitate, and the Tris buffer solution contains 10mM EDTA and 5mg / ml sodium deoxycholate.

[0014] Further, in step (2), the high molecular material is polylactic acid-glycolic acid, the organic solvent is ethyl acetate; the drug is an imaging agent, the mass ratio of the imaging agent to the high molecular material is 0.02:1-0.2:1, the imaging agent dissolved in the oil phase is any one of DiR, IR780 and SPIO, and the imaging agent dissolved in the water phase is doxorubicin.

[0015] Further, in step (2), the external water phase is 2.5% polyvinyl alcohol or vitamin polyethylene glycol succinate solution; the volatile water phase is 0.3% polyvinyl alcohol or vitamin polyethylene glycol succinate solution.

[0016] Further, in step (2), the centrifugation temperature is 4℃, the speed is 30000-35000rpm, and the time is 20min.

[0017] Further, in step (3), the mass ratio of the E. coli DH5ɑ modified outer membrane to the final precipitated nano-carrier is 1:5, the ultrasonic time is 3min, and the extrusion times are 11.

[0018] The third technical solution of the application is:

[0019] The application of the brain-targeting drug delivery system based on the modified E. coli DH5ɑ outer membrane in the preparation of a drug preparation for targeting brain endothelial cells.

[0020] The application provides a brain-targeting drug delivery system based on an outer membrane of engineered Escherichia coli DH5a and a preparation method and application thereof, and has the beneficial effects that: the method is simple, raw materials are easy to obtain, is suitable for research and application transformation, the brain-targeting drug delivery system can penetrate the blood-brain barrier, is novel, low in cost, high in efficiency, can play a good brain-targeting role, is highly operable, novel and economic. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 A schematic diagram for construction and in-vivo behavior of the brain-targeting drug delivery system based on the outer membrane of the engineered Escherichia coli DH5a according to the application;

[0022] Figure 2 A characterization diagram of the outer membrane of the Escherichia coli DH5a with lipopolysaccharide and the outer membrane structure of the Escherichia coli DH5a by transmission electron microscopy;

[0023] Figure 3 A whole protein distribution diagram of the outer membrane of the Escherichia coli DH5a with lipopolysaccharide and the outer membrane of the Escherichia coli DH5a by SDS-PAGE;

[0024] Figure 4 An outer membrane protein A expression diagram of the outer membrane of the Escherichia coli DH5a with lipopolysaccharide and the outer membrane of the Escherichia coli DH5a by western blot;

[0025] Figure 5 A hemolytic level diagram of the outer membrane of the Escherichia coli DH5a with lipopolysaccharide, the outer membrane of the Escherichia coli DH5a, red blood cell membrane and lipopolysaccharide by a hemolytic experiment;

[0026] Figure 6 A characterization diagram of the outer membrane nanocarrier structure of the Escherichia coli DH5a by transmission electron microscopy;

[0027] Figure 7 A particle size diagram of the outer membrane nanocarrier of the Escherichia coli DH5a by dynamic light scattering;

[0028] Figure 8 An outer membrane protein A expression diagram of the outer membrane nanocarrier of the Escherichia coli DH5a, the drug delivery system of the outer membrane of the Escherichia coli DH5a and the blank nanocarrier by western blot;

[0029] Figure 9 A hemolytic level diagram of the outer membrane nanocarrier of the Escherichia coli DH5a with lipopolysaccharide, the outer membrane nanocarrier of the Escherichia coli DH5a, red blood cell membrane and lipopolysaccharide by a hemolytic experiment;

[0030] Figure 10The uptake profile of various E. coli DH5α outer membrane nanocarriers loaded with doxorubicin (the concentration of doxorubicin was 5 μg / mL, and the administration time was 3 h) in brain microvascular endothelial cells for quantitative characterization;

[0031] Figure 11 The uptake profile of E. coli DH5α outer membrane nanocarriers loaded with doxorubicin after treatment with different contents of exogenous outer membrane protein A protein (the concentration of doxorubicin was 5 μg / mL, and the administration time was 1.5 h) in brain microvascular endothelial cells for quantitative characterization;

[0032] Figure 12 The uptake profile of E. coli DH5α outer membrane nanocarriers loaded with doxorubicin after treatment with different contents of gp96 antibody (the concentration of doxorubicin was 5 μg / mL, and the administration time was 1.5 h) in brain microvascular endothelial cells for quantitative characterization;

[0033] Figure 13 The expression profile of gp96 on brain microvascular endothelial cells bEnd.3 up-regulated by E. coli DH5α outer membrane, E. coli outer membrane DH5α nanocarriers, and E. coli outer membrane DH5α drug-loaded systems characterized by western blot;

[0034] Figure 14 The uptake profile of E. coli DH5α outer membrane nanocarriers loaded with doxorubicin after treatment with different endocytosis inhibitors (the concentration of doxorubicin was 5 μg / mL, and the administration time was 1.5 h) in brain microvascular endothelial cells for quantitative characterization;

[0035] Figure 15 The accumulation profile of IR780 at different times in normal mouse brains treated with IR780-loaded E. coli DH5α outer membrane nanocarriers, wherein the dose of IR780 was 2.5 μg per mouse, and the administration time was 4 h, 12 h, and 24 h, for quantitative characterization;

[0036] Figure 16 The accumulation profile of iron elements in normal mouse brains treated with various SPIO-loaded membrane nanocarrier systems, wherein the dose of SPIO was 5 mg / kg of mice, and the administration time was 8 h, for quantitative characterization by inductively coupled plasma-mass spectrometry;

[0037] Figure 17 The accumulation profile of IR780 at different times in normal mouse brains treated with various IR780-loaded nanocarrier systems, wherein the dose of IR780 was 2.5 μg per mouse, and the administration time was 2 h, 4 h, and 8 h, for qualitative characterization by small animal imaging;

[0038] Figure 18Distribution of doxorubicin-loaded red blood cell membrane nanocarriers, Angiopep 2 modified nanoparticles and E. coli DH5a outer membrane nanocarriers in different brain regions of normal mice, wherein the dose of doxorubicin is 5 mg / kg of mice, and the injection is performed twice with an interval of 12 h, and the perfusion is performed 12 h after the second injection.

[0039] Figure 19 mRNA relative expression levels of proinflammatory factors TNF-α, IL-6 and IL-1β on normal mouse brains treated with physiological saline, E. coli DH5a outer membrane drug delivery system and lipopolysaccharide, which are quantitatively characterized by real-time fluorescent quantitative PCR method;

[0040] Figure 20 Concentrations of proinflammatory factors TNF-α, IL-6 and IL-1β on normal mouse brains treated with physiological saline, E. coli DH5a outer membrane drug delivery system and lipopolysaccharide, which are quantitatively characterized by enzyme-linked immunosorbent assay method;

[0041] Figure 21 Concentrations of proinflammatory factors TNF-α, IL-6 and IL-1β in normal mouse serum treated with physiological saline, E. coli DH5a outer membrane drug delivery system and lipopolysaccharide, which are quantitatively characterized by enzyme-linked immunosorbent assay method;

[0042] Figure 22 Concentrations of proinflammatory factors TNF-α, IL-6 and IL-1β in normal mouse liver treated with physiological saline, E. coli DH5a outer membrane drug delivery system and lipopolysaccharide, which are quantitatively characterized by enzyme-linked immunosorbent assay method;

[0043] Figure 23 Concentrations of proinflammatory factors TNF-α, IL-6 and IL-1β in normal mouse spleen treated with physiological saline, E. coli DH5a outer membrane drug delivery system and lipopolysaccharide, which are quantitatively characterized by enzyme-linked immunosorbent assay method;

[0044] Figure 24 Concentrations of liver function indicators glutathione pyrotransaminase and glutathione transaminase and kidney function indicators urea nitrogen and creatinine in normal mouse serum treated with physiological saline, E. coli DH5a outer membrane drug delivery system and lipopolysaccharide, which are quantitatively characterized by kit method;

[0045] Figure 25Figure 2 is a hematoxylin-eosin staining method for characterizing the physiological saline, E. coli DH5α outer membrane drug delivery system, lipopolysaccharide E. coli DH5α outer membrane drug delivery system, and lipopolysaccharide treatment on organ histology and morphology observation chart, the scale in the figure is 100 μm. DETAILED DESCRIPTION

[0046] Inspired by the fact that the outer membrane of bacteria can specifically bind and invade BBB endothelial cells due to carrying outer membrane proteins, the present application utilizes the BBB invasion ability of E. coli DH5α outer membrane to apply to brain-targeted drug delivery, and constructs a brain-targeted drug delivery system based on the E. coli DH5α modified outer membrane capable of penetrating the blood-brain barrier.

[0047] The present application provides a brain-targeted drug delivery system based on the modified E. coli DH5α outer membrane, which selects a biodegradable polymer material as a basic carrier, and uses the ultrasonic emulsification-solvent evaporation method to prepare a nano-carrier loaded with drugs inside, and the nano-carrier surface is wrapped with E. coli DH5α modified outer membrane with brain-targeted invasion ability and lipopolysaccharide removal, thereby constructing a brain-targeted drug delivery system based on the modified E. coli DH5α outer membrane. This approach mainly relies on the interaction between outer membrane protein A and up-regulated gp96 on BBB endothelial cells to mediate transcellular action, and this biomimetic nano-engineering strategy greatly improves the brain-targeting efficiency of the drug delivery system based on the biomimetic system while ensuring low toxicity and biocompatibility.

[0048] The preparation method of the above-mentioned brain-targeted drug delivery system based on the modified E. coli DH5α outer membrane is as follows:

[0049] Step one, extraction of E. coli DH5α modified outer membrane;

[0050] Step two, preparation of nano-carrier;

[0051] Step three, preparation of E. coli DH5α modified outer membrane nano-carrier.

[0052] The preparation method of the above-mentioned brain-targeted drug delivery system based on the modified E. coli DH5α outer membrane can be used for preparing drug preparations targeting brain endothelial cells.

[0053] The above-mentioned brain-targeted drug delivery system based on the E. coli DH5α modified outer membrane capable of penetrating the blood-brain barrier is described in detail in the specification. Figure 1 , Figure 1 The construction and in vivo behavior of the brain-targeted drug delivery system based on the modified E. coli DH5α outer membrane according to the present application are shown in the schematic diagram of Figure 1As shown, the brain-targeting drug delivery system based on the outer membrane of the modified E. coli DH5ɑ utilizes the outer membrane of the DH5ɑ to target the brain microvascular endothelial cells, and in the blood vessels of the brain, the system penetrates the BBB through the specific recognition and binding between the outer membrane protein A and gp96, and is further distributed in the intracranial interstitial space.

[0054] In order to make the above-mentioned purposes, features and advantages of the present application more obvious and easy to understand, the technical solutions of the present application will be further described below in combination with embodiments. However, the present application is not limited to the listed embodiments, and any known changes within the scope of the claimed rights of the present application should also be included.

[0055] The term "one embodiment" or "an embodiment" as used herein means that a particular feature, structure, or characteristic described can be included in at least one implementation of the present application. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are they necessarily all referring to a single, alternative embodiment, or a single implementation.

[0056] Embodiment 1

[0057] The present embodiment demonstrates a preparation method of a brain-targeting drug delivery system based on the outer membrane of the modified E. coli DH5ɑ as follows:

[0058] 1. Extracting the modified outer membrane of E. coli DH5ɑ: overnight culture E. coli DH5ɑ in pH 7.4 LB medium to make its OD 600 value reach 1.5, centrifugal collection of E. coli precipitate, centrifugal parameters are 4℃, 5000g, 10min; resuspend the precipitate in four times the volume of pH 7.4 phosphate buffer, 300W ultrasonic crushing for 30min; centrifugal the crushed bacterial liquid at 4℃, 2900g for 1h; add 7.5 times the volume of 0.1M Tris buffer (containing 10mM EDTA and 5mg / ml sodium deoxycholate) to the precipitate, 4℃, 20000g, 1h high-speed centrifugation; collect the supernatant, 4℃, 125000-150000g, 2h high-speed centrifugation twice; disperse the final E. coli DH5ɑ modified outer membrane precipitate in water and store at -20℃ for later use.

[0059] 2. Preparation of nanocarriers: 30 mg of polylactic acid-glycolic acid was weighed in 1 mL of ethyl acetate as oil phase, 100 μl of water was added dropwise into the vortexed oil phase, and ultrasonic emulsification was performed to form a water-in-oil emulsion; the emulsion was added dropwise into 2 mL of 2.5% polyvinyl alcohol solution which was vortexed, and ultrasonic emulsification was performed to form a water-in-oil-in-water double emulsion; the double emulsion was quickly poured into 50 mL of 0.3% polyvinyl alcohol solution, and stirred overnight to remove ethyl acetate by volatilization; the obtained nanocarrier suspension was purified by high-speed centrifugation at 3000-35000 rpm for 20 min; the purified non-wrapped nanocarriers were ultrasonically dispersed in an appropriate amount of ultrapure water, and high-speed centrifugation was performed for water washing twice; and the final precipitated nanocarriers were dispersed in ultrapure water.

[0060] 3. The obtained E. coli DH5ɑ modified outer membrane was ultrasonicated for 30 s, mixed with the final nanocarriers according to a mass ratio of 1:5, and ultrasonicated, and then extruded 11 times using a 200 nm polycarbonate membrane to obtain a brain-targeting drug delivery system based on E. coli DH5ɑ modified outer membrane, which was freshly prepared for use.

[0061] Example 2

[0062] In order to investigate the E. coli DH5ɑ outer membrane extracted by the present scheme, the outer membrane structure was investigated by transmission electron microscopy. Please refer to Figure 2 , Figure 2 The figure is a transmission electron microscopy characterization of the lipopolysaccharide-containing E. coli DH5ɑ outer membrane and the E. coli DH5ɑ outer membrane structure. As shown in Figure 2 , the transmission electron microscopy results show that the lipopolysaccharide-containing E. coli DH5ɑ outer membrane and the E. coli DH5ɑ outer membrane are successfully extracted by the present scheme.

[0063] Example 3

[0064] In order to investigate the protein expression of the E. coli outer membrane extracted by the present scheme, the lipopolysaccharide-containing E. coli DH5ɑ outer membrane and the E. coli DH5ɑ outer membrane were prepared, and the outer membrane protein expression was investigated by SDS-PAGE. Please refer to Figure 3 , Figure 3 The figure is a full protein distribution of the lipopolysaccharide-containing E. coli DH5ɑ outer membrane and the E. coli DH5ɑ outer membrane characterized by SDS-PAGE. As shown in Figure 3 , the SDS-PAGE results show the protein differences of the lipopolysaccharide-containing E. coli DH5ɑ outer membrane and the E. coli DH5ɑ outer membrane due to different extraction methods.

[0065] Example 4

[0066] To investigate the expression of outer membrane protein A of the nanosystem constructed in this scheme, outer membrane protein A with MBP tag, outer membrane of E. coli DH5ɑ with lipopolysaccharide and outer membrane of E. coli DH5ɑ were prepared. Please refer to Figure 4 , Figure 4 To characterize the outer membrane protein A expression of outer membrane of E. coli DH5ɑ with lipopolysaccharide and outer membrane of E. coli DH5ɑ by western blot. As shown in Figure 4 , the western blot results showed that there were outer membrane protein A with the ability to cross the blood-brain barrier on the outer membrane of E. coli DH5ɑ with lipopolysaccharide and outer membrane of E. coli DH5ɑ.

[0067] Example 5

[0068] To investigate the hemolysis phenomenon of E. coli outer membrane, outer membrane of E. coli DH5ɑ with lipopolysaccharide, outer membrane of E. coli DH5ɑ and red blood cell membrane were detected by hemolysis experiment to detect the hemolysis level of the above-mentioned protein. Please refer to Figure 5 , Figure 5 To detect the hemolysis level of outer membrane of E. coli DH5ɑ with lipopolysaccharide, outer membrane of E. coli DH5ɑ, red blood cell membrane and lipopolysaccharide by hemolysis experiment. As shown in Figure 5 , the results showed that lipopolysaccharide and outer membrane of E. coli DH5ɑ with lipopolysaccharide could cause hemolysis, but no obvious hemolysis phenomenon was detected in outer membrane of E. coli DH5ɑ and red blood cell membrane.

[0069] Example 6

[0070] To investigate the structure of the nanocarrier system constructed in this scheme, E. coli DH5ɑ outer membrane nanocarrier was prepared, and the structure of the carrier system was investigated by transmission electron microscopy. Please refer to Figure 6 , Figure 6 To characterize the structure of E. coli DH5ɑ outer membrane nanocarrier by transmission electron microscopy. As shown in Figure 6 , the transmission electron microscopy results showed that the nanocarrier system with shell-core structure was successfully constructed in this scheme.

[0071] Example 7

[0072] To investigate the particle size and uniformity of the nanocarrier system constructed in this scheme. Please refer to Figure 7 , Figure 7 To characterize the particle size of E. coli DH5ɑ outer membrane nanocarrier by dynamic light scattering method. As shown in Figure 7 , the particle size of E. coli DH5ɑ outer membrane nanocarrier was 115 nm, and the polydispersity coefficient (PDI) was 0.241, indicating that the constructed nanocarrier system was uniform and stable.

[0073] Example 8

[0074] To investigate whether the outer membrane protein A is expressed in the nanocarrier system constructed in this scheme, the outer membrane nanocarrier of E. coli DH5a, the outer membrane drug-loaded system of E. coli DH5a, and the blank nanocarrier are prepared. By western blot, it is investigated that the outer membrane protein A is transferred into the nanocarrier system by the means of extrusion wrapping (mentioned in Example 1). Please refer to Figure 8 , Figure 8 To characterize the expression of outer membrane protein A in the outer membrane nanocarrier of E. coli DH5a, the outer membrane drug-loaded system of E. coli DH5a, and the blank nanocarrier by western blot. As shown in Figure 8 , the western blot result shows that the outer membrane protein A of the outer membrane nanocarrier system of E. coli DH5a can mediate the penetration through the blood-brain barrier.

[0075] Example 9

[0076] To investigate the hemolysis phenomenon of the outer membrane nanocarrier system of E. coli DH5a, the lipopolysaccharide-containing outer membrane nanocarrier of E. coli DH5a, the outer membrane nanocarrier of E. coli DH5a, and the red blood cell membrane nanocarrier are detected by hemolysis experiment to detect the hemolysis level of the above nanosystems. Please refer to Figure 9 , Figure 9 To detect the hemolysis level of the lipopolysaccharide-containing outer membrane nanocarrier of E. coli DH5a, the outer membrane nanocarrier of E. coli DH5a, and the red blood cell membrane, and lipopolysaccharide by hemolytic experiment. As shown in Figure 9 , the results show that lipopolysaccharide and lipopolysaccharide-containing outer membrane nanocarrier of E. coli DH5a can cause hemolysis, but no obvious hemolysis phenomenon is detected in the outer membrane nanocarrier of E. coli DH5a and the red blood cell membrane nanocarrier.

[0077] Example 10

[0078] To investigate the uptake of doxorubicin-loaded nanocarrier systems in brain microvascular endothelial cells, mouse brain endothelial cells (bEND.3) are inoculated in a 6-well plate, and unmodified nanocarriers, red blood cell membrane nanocarriers, and outer membrane nanocarriers of E. coli DH5a are added respectively for 3h incubation. The concentration of doxorubicin is 5μg / mL, and the cell uptake is detected by flow cytometry FL2 channel. The conclusion of this example is shown in Figure 10 , Figure 10 To quantitatively characterize the uptake of doxorubicin-loaded nanocarriers (the concentration of doxorubicin is 5μg / mL, and the administration time is 3h) in brain microvascular endothelial cells. As shown in Figure 10As shown, compared with unmodified nanocarriers and erythrocyte membrane nanocarriers, the E. coli DH5α outer membrane nanocarrier was taken up more by bEND.3 cells, indicating that it is more likely to accumulate in endothelial cells.

[0079] Example 11

[0080] To investigate the uptake of the *E. coli* DH5α outer membrane nanocarrier on brain microvascular endothelial cells after treatment with exogenous outer membrane protein A, mouse brain endothelial cells (bEND.3) were seeded in 6-well plates. Pretreatment with exogenous outer membrane protein A (without it), 10 μg, 20 μg, and 50 μg) for 1.5 h was performed. Then, the cells were co-incubated with the doxorubicin-loaded DH5α outer membrane nanocarrier system (5 μg / mL) for 1.5 h. Cell uptake was detected by flow cytometry using the FL2 channel. The conclusions of this example can be found in [link to relevant documentation]. Figure 11 , Figure 11 This image shows the quantitative uptake of doxorubicin-loaded *E. coli* DH5α outer membrane nanocarriers (doxorubicin concentration 5 μg / mL, administration time 1.5 h) on brain microvascular endothelial cells after treatment with different amounts of exogenous outer membrane protein A. Figure 11 As shown, exogenous protein A can inhibit the uptake of the DH5α outer membrane nanocarrier system by endothelial cells, up to 18%, confirming that outer membrane protein A is involved in the uptake of the detoxified bacterial outer membrane-encapsulated nanocarrier by brain endothelial cells. Combined with Case 11, this shows that the specific recognition between outer membrane protein A and gp96 is closely involved in the uptake of the DH5α outer membrane nanocarrier system by blood-brain barrier endothelial cells.

[0081] Example 12

[0082] To investigate the uptake of the DH5α outer membrane nanocarrier system on brain microvascular endothelial cells after treatment with gp96 antibody, mouse brain endothelial cells (bEND.3) were seeded in 6-well plates. Pretreatment with gp96 antibody-free, 0.24 μg gp96 antibody, and 1.2 μg gp96 antibody for 1.5 h was performed, followed by incubation with the DH5α outer membrane nanocarrier system loaded with doxorubicin and the different antibody doses for 1.5 h. The doxorubicin concentration was 5 μg / mL. Cell uptake was detected by flow cytometry using the FL2 channel. The conclusions of this example can be found in [link to relevant documentation]. Figure 12 , Figure 12 This image shows the quantitative uptake of doxorubicin-loaded E. coli DH5α outer membrane nanocarriers (doxorubicin concentration 5 μg / mL, administration time 1.5 h) on brain microvascular endothelial cells after treatment with different concentrations of gp96 antibody. Figure 12As shown in Figure 13, gp96 antibody can inhibit the uptake of DH5a outer membrane nanocarrier system by endothelial cells, with a maximum inhibition of 20%, confirming that gp96 antibody is involved in the uptake of DH5a outer membrane nanocarrier system by brain endothelial cells. In combination with Example 13, it is shown that the E. coli DH5a outer membrane nanocarrier system crosses the blood-brain barrier through specific recognition between outer membrane protein A and gp96.

[0083] Example 13

[0084] In order to investigate the upregulation of gp96 on brain microvascular endothelial cells caused by E. coli DH5a outer membrane, E. coli DH5a outer membrane nanocarrier and E. coli DH5a outer membrane nanocarrier drug system, mouse brain endothelial cells (bEND.3) were inoculated in T25 culture bottles, and DH5a outer membrane, DH5a outer membrane nanocarrier and DH5a outer membrane drug system were added respectively, with a protein concentration of 50 μg / ml, and treated for 24 h. After obtaining the cell sample, the expression of gp96 on the endothelial cells was investigated by denaturing quantitative western blot. The conclusion of this example can be seen in Figure 13 , Figure 13 Figure 14 is a diagram showing the upregulation of gp96 expression on endothelial cells bEnd.3 by E. coli DH5a outer membrane, E. coli outer membrane DH5a nanocarrier and E. coli outer membrane DH5a drug system, as shown in Figure 13 the western blot results show that DH5a outer membrane, DH5a outer membrane nanocarrier and DH5a outer membrane drug system can upregulate gp96 protein on bEnd.3, in combination with Example 13, it is shown that the E. coli DH5a outer membrane nanocarrier system crosses the blood-brain barrier through the interaction between outer membrane protein A and gp96.

[0085] Example 14

[0086] In order to investigate the endocytosis pathway of E. coli DH5a outer membrane nanocarrier system on brain microvascular endothelial cells, mouse brain endothelial cells (bEND.3) were inoculated in a 6-well plate, and first pretreated with no endocytosis pathway inhibitor, clorgyline 10 μg / ml for clathrin pathway inhibitor, nystatin 25 μg / ml for caveolin pathway inhibitor, methyl-β-cyclodextrin 6.5 mg / ml for lipid raft pathway inhibitor, amiloride 100 μg / ml for macropinocytosis pathway inhibitor for 1.5 h, then E. coli DH5a outer membrane nanocarrier system loaded with doxorubicin and the above inhibitors were added simultaneously and incubated for 1.5 h, with a concentration of doxorubicin of 5 μg / mL, and the cell uptake was detected by flow cytometry FL2 channel. The conclusion of this example can be seen in Figure 14 , Figure 14Figure 6. Quantitative characterization of the uptake of doxorubicin-loaded E. coli DH5a OM nanocarriers (5 μg / mL of doxorubicin, 1.5 h of administration time) in brain microvascular endothelial cells after treatment with different endocytosis inhibitors. As shown in Figure 14

[0087] Example 15

[0088] To quantitatively investigate the accumulation of IR780 in normal mouse brain at different time points after treatment with IR780-loaded nanocarriers, normal mice were injected with IR780-loaded E. coli DH5a OM nanocarriers via the tail vein. Four hours, 12 hours and 24 hours after injection, the mice were perfused and fixed, and the brain tissue was removed and quantitatively analyzed for IR780 accumulation using a microplate reader. The dose of IR780 was 2.5 μg per mouse. The results of this example are shown in Figure 5. Figure 15 Figure 15 Figure 5. Quantitative characterization of the accumulation of IR780 in normal mouse brain at different time points after treatment with IR780-loaded E. coli DH5a OM nanocarriers. As shown in Figure 15

[0089] Most nanocarriers, even those designed to enhance BBB penetration, have low brain accumulation (<1% dose / g-brain) after intravenous administration. Therefore, the accumulation of 1.23% dose / g-brain achieved after administration is very attractive, as it is significantly higher than the accumulation of most other carrier systems reported to date that use receptor-mediated transcytosis strategies.

[0090] Example 16

[0091] ​​​To quantitatively investigate the accumulation of iron in SPIO in the brains of normal mice treated with various SPIO-loaded nanomedicine delivery systems at different time points, normal mice were injected intravenously with saline, erythrocyte membrane nanocarriers, Angiopep 2-modified nanocarriers, DH5α outer membrane nanocarrier systems (without pre-administration of gp96 antibody), and DH5α outer membrane nanocarrier systems (pre-administered with gp96 antibody), respectively. Eight hours after injection, the mice were perfused, and brain tissue was collected. Inductively coupled plasma mass spectrometry (ICP-MS) was used to quantitatively observe the accumulation of iron in the nanocarriers within the normal brain tissue. The SPIO dose was 5 mg / kg of mice. For the conclusions of this example, please refer to [link to relevant documentation]. Figure 16 , Figure 16 This image shows the accumulation of iron in normal mouse brains treated with various SPIO-supported membrane nanocarrier systems, as quantitatively characterized by inductively coupled plasma mass spectrometry (ICP-MS). Figure 16 As shown, the E. coli DH5α outer membrane nanocarrier system accumulated the most nanocarriers in mouse brain tissue, and pretreatment with gp96 antibody significantly reduced the accumulation of nanocarriers in mouse brain tissue, further demonstrating the role of gp96-mediated transcellular transport in the blood-brain barrier crossing behavior of this nanocarrier system.

[0092] Example 17

[0093] To qualitatively examine drug accumulation in the brains of normal mice treated with various nanodelivery systems carrying IR780 at different time points, normal mice were injected intravenously with IR780 via tail veins using erythrocyte membrane nanocarriers and E. coli DH5α outer membrane nanocarrier systems, respectively. Nanoparticle accumulation in the brains of live mice was observed using a small animal imaging system at 2 h, 4 h, and 8 h. The dose of IR780 was 2.5 μg per mouse. For the conclusions of this example, please refer to [link to relevant documentation]. Figure 17 , Figure 17 Accumulation maps of IR780 at different time points in normal mouse brains treated with various nanocarrier systems carrying IR780, for qualitative characterization by imaging techniques in small animals. (Example:) Figure 17 As shown, the erythrocyte membrane nanocarriers did not accumulate significantly in mice. The E. coli DH5α outer membrane nanocarrier system showed the highest accumulation of nanoparticles in mouse brain tissue, indicating that it has optimal blood-brain barrier penetration capability.

[0094] Example 18

[0095] In order to investigate the distribution of various doxorubicin-loaded nanodelivery systems in different brain regions of normal mice, the normal mice were injected with red blood cell membrane nanocarriers, Angiopep 2 modified nanoparticles and E. coli DH5ɑ outer membrane nanocarrier systems via the tail vein, a total of two injections with an interval of 12 h, and perfusion was performed 12 h after the second injection. The mouse brain tissues were taken, dehydrated with sucrose, frozen sectioned, and the distribution of nanocarrier systems in different brain regions of normal brain tissues was observed by confocal microscopy. The dose of doxorubicin injected each time was 5 mg / kg of mice. The conclusion of this example can be seen in Figure 18 , Figure 18 The distribution of doxorubicin-loaded red blood cell membrane nanocarriers, Angiopep 2 modified nanoparticles and E. coli DH5ɑ outer membrane nanocarriers in different brain regions of normal mice characterized by confocal microscopy is shown in FIG. 3. As shown in FIG. 3, compared with Angiopep 2 modified nanocarriers and red blood cell membrane nanocarriers, the E. coli DH5ɑ outer membrane nanocarrier system was taken up more in each brain region. Figure 18

[0096] Example 19

[0097] In order to quantitatively investigate the mRNA relative expression levels of proinflammatory factors TNF-α, IL-6 and IL-1β in normal mouse brains treated with physiological saline, E. coli DH5ɑ outer membrane drug-loaded systems and lipopolysaccharide-containing E. coli DH5ɑ outer membrane drug-loaded systems, the normal mice were injected with the above nanocarrier systems via the tail vein. Perfusion was performed 24 h after injection, and the mouse brain tissues were taken. The expression of proinflammatory factors TNF-α, IL-6 and IL-1β was quantitatively detected by real-time fluorescent quantitative PCR. The dose of protein was 6.86 mg / kg. The conclusion of this example can be seen in Figure 19 , Figure 19 The mRNA relative expression levels of proinflammatory factors TNF-α, IL-6 and IL-1β in normal mouse brains treated with physiological saline, E. coli DH5ɑ outer membrane drug-loaded systems and lipopolysaccharide-containing E. coli DH5ɑ outer membrane drug-loaded systems quantitatively characterized by real-time fluorescent quantitative PCR are shown in FIG. 4. As shown in FIG. 4, at the mRNA level, the lipopolysaccharide-containing E. coli DH5ɑ outer membrane drug-loaded system significantly increased the production of intracranial TNF-α, IL-1β and IL-6, while the E. coli DH5ɑ outer membrane drug-loaded system did not cause any obvious inflammatory changes, indicating that the nanodelivery system has good biological safety. Figure 19

[0098] Example 20

[0099] ​​In order to quantitatively investigate the protein levels of proinflammatory factors TNF-α, IL-6 and IL-1β in physiological saline, E. coli DH5α outer membrane drug delivery system, E. coli DH5α outer membrane drug delivery system with lipopolysaccharide, and normal mouse brain treated with lipopolysaccharide, the above various nano-carrier systems were injected into the tail vein of normal mice, perfused 24 h after injection, and the mouse brain tissue was taken. The protein expression levels of proinflammatory factors TNF-α, IL-6 and IL-1β in the brain tissue and serum were quantitatively detected by enzyme-linked immunosorbent assay. The protein dose was 6.86 mg / kg, and the lipopolysaccharide dose was 0.823 μg / kg mouse. The conclusion of this example is referred to Figures 20-23 , Figure 20 The concentration diagram of proinflammatory factors TNF-α, IL-6, and IL-1β in physiological saline, E. coli DH5α outer membrane drug delivery system, E. coli DH5α outer membrane drug delivery system with lipopolysaccharide, and normal mouse brain treated with lipopolysaccharide quantitatively characterized by enzyme-linked immunosorbent assay; Figure 21 The concentration diagram of proinflammatory factors TNF-α, IL-6, and IL-1β in physiological saline, E. coli DH5α outer membrane drug delivery system, E. coli DH5α outer membrane drug delivery system with lipopolysaccharide, and normal mouse serum treated with lipopolysaccharide quantitatively characterized by enzyme-linked immunosorbent assay; Figure 22 The concentration diagram of proinflammatory factors TNF-α, IL-6, and IL-1β in physiological saline, E. coli DH5α outer membrane drug delivery system, E. coli DH5α outer membrane drug delivery system with lipopolysaccharide, and normal mouse liver treated with lipopolysaccharide quantitatively characterized by enzyme-linked immunosorbent assay; Figure 23 The concentration diagram of proinflammatory factors TNF-α, IL-6, and IL-1β in physiological saline, E. coli DH5α outer membrane drug delivery system, E. coli DH5α outer membrane drug delivery system with lipopolysaccharide, and normal mouse spleen treated with lipopolysaccharide quantitatively characterized by enzyme-linked immunosorbent assay. As shown in Figures 20-23 lipopolysaccharide, E. coli DH5α outer membrane drug delivery system with lipopolysaccharide significantly increased the production of intracranial TNF-α and IL-6 at the protein level, while E. coli DH5α outer membrane drug delivery system did not cause any obvious inflammatory changes, indicating that the improved nano-carrier system has good biological safety.

[0100] Example 21

[0101] In order to quantitatively investigate the concentrations of liver function indicators glutamic-pyruvic transaminase and glutamic-oxalacetic transaminase and kidney function indicators urea nitrogen and creatinine in the normal mouse serum treated by normal saline, E. coli DH5a outer membrane drug delivery system and E. coli DH5a outer membrane drug delivery system with lipopolysaccharide, the normal mice were injected with the above-mentioned nano-carrier systems through the tail vein, perfused 24 hours after injection, and the mouse serum was taken to quantitatively detect the concentrations of liver function indicators glutamic-pyruvic transaminase and glutamic-oxalacetic transaminase and kidney function indicators urea nitrogen and creatinine in the normal mouse serum by kit method. The dose of IR780 was 2.5 μg per mouse. The conclusions of this example can be seen in Figure 24 , Figure 24 The figure of the concentrations of liver function indicators glutamic-pyruvic transaminase and glutamic-oxalacetic transaminase and kidney function indicators urea nitrogen and creatinine in the normal mouse serum treated by normal saline, E. coli DH5a outer membrane drug delivery system and E. coli DH5a outer membrane drug delivery system with lipopolysaccharide, which were quantitatively characterized by kit method. As shown in Figure 24 , the E. coli DH5a outer membrane drug delivery system with lipopolysaccharide significantly increased the production of kidney function indicators, while the E. coli DH5a outer membrane drug delivery system did not cause any obvious change, indicating that it did not cause liver and kidney function damage, which shows that the improved nano-carrier system has good biological safety.

[0102] Example 22

[0103] In order to investigate whether the tissues and organs will change after being treated by normal saline, E. coli DH5a outer membrane drug delivery system, E. coli DH5a outer membrane drug delivery system with lipopolysaccharide and lipopolysaccharide, the normal mice were injected with the above-mentioned nano-carrier systems through the tail vein, and the protein was administered at a dose of 6.86 mg / kg, and the lipopolysaccharide was administered at a dose of 0.823 μg / kg mouse. Three times of drug administration were given per week, respectively on the first, fourth and sixth days. 24 hours after the last administration, the mice were perfused, and the mouse organs were taken out, then the mouse organs were embedded and sectioned and stained with hematoxylin-eosin. The conclusions of this example can be seen in Figure 25 , the scale is 100 μm. Figure 25 The figure of the observation of histology and morphology of organs treated by normal saline, E. coli DH5a outer membrane drug delivery system, E. coli DH5a outer membrane drug delivery system with lipopolysaccharide and lipopolysaccharide by hematoxylin-eosin staining method. As shown in Figure 25 , the E. coli DH5a outer membrane drug delivery system with lipopolysaccharide and lipopolysaccharide will cause slight changes in the morphology of the lung, liver and kidney, indicating that it will cause a certain degree of inflammatory reaction, while the E. coli DH5a outer membrane drug delivery system does not cause any obvious change, which shows that the improved nano-carrier system has good biological safety.

[0104] Compared with the prior art, the brain-targeting drug delivery system based on the outer membrane of the modified E. coli DH5a, the preparation method and the application have the following beneficial effects: the drug delivery system is based on the interaction between the outer membrane protein A of the outer membrane of the E. coli DH5a and gp96 on the endothelial cells of the blood-brain barrier, and mediates the drug delivery system in the blood circulation to cross the barrier and finally enter the intracranial interstitium. The method is simple, has good biological safety and superior delivery efficiency, and has operability and considerable economic benefits.

[0105] It should be noted that the above examples are only used to illustrate the technical solutions of the present application and are not limiting. Although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present application can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present application, and they should be covered in the scope of the claims of the present application.

Claims

1. A brain-targeting drug delivery system based on the outer membrane of engineered Escherichia coli DH5α, characterized in that: The application discloses a brain-targeting drug delivery system, which comprises a biodegradable polymer material, an outer membrane of Escherichia coli DH5a and an imaging agent. 2.The brain-targeting drug delivery system based on the outer membrane of engineered Escherichia coli DH5α according to claim 1, wherein, The preparation method comprises the following steps: (1) extracting the outer membrane of Escherichia coli DH5a: culturing Escherichia coli DH5a in a culture medium overnight, so that the OD 600 value reaches 1.5, centrifuging to collect the Escherichia coli DH5a bacterial body precipitate, resuspending the bacterial body precipitate in a phosphate buffer, and crushing the bacterial body by ultrasonic waves to obtain a crushed bacterial liquid; centrifuging the crushed bacterial liquid to obtain a precipitate; adding a Tris buffer into the precipitate, and high-speed centrifuging to collect a supernatant; high-speed centrifuging the supernatant again to obtain the outer membrane of Escherichia coli DH5a with removed lipopolysaccharide; and dispersing the outer membrane of Escherichia coli DH5a in ultrapure water for storage; (2) preparing a nano carrier: weighing the polymer material, dissolving the polymer material in an organic solvent to form an oil phase; dissolving the drug in the oil phase to form part of the oil phase, and using water itself as a water phase, or dissolving the drug in water to form a water phase; adding the water phase into the oil phase under vortex in drops, and ultrasonically emulsifying to form a water-in-oil emulsion; adding the water-in-oil emulsion into an outer water phase under vortex in drops, and ultrasonically emulsifying to form a water-in-oil-in-water double emulsion; rapidly pouring the double emulsion into a volatile water phase, stirring overnight to volatilize, and forming a nano carrier suspension; high-speed centrifuging the nano carrier suspension to purify the nano carrier suspension, and obtaining a nano carrier precipitate; ultrasonically dispersing the nano carrier precipitate in water, high-speed centrifuging, and obtaining a final nano carrier precipitate; and dispersing the final nano carrier precipitate in ultrapure water; (3) preparing the nano carrier coated with the outer membrane of Escherichia coli DH5a: ultrasonically dispersing the outer membrane of Escherichia coli DH5a in water, mixing the outer membrane of Escherichia coli DH5a with the final nano carrier precipitate, and ultrasonically dispersing the mixture, so as to obtain the brain-targeting drug delivery system based on the outer membrane of the modified Escherichia coli DH5a. 3.The brain-targeting drug delivery system based on the outer membrane of engineered E. coli DH5α according to claim 2, characterized in that: In step (1), the culture medium is a pH 7.4 LB culture medium, and the volume ratio of the bacterial body precipitate to the phosphate buffer is 1:4, and the pH value of the phosphate buffer is 7.

4. 4.The brain-targeting drug delivery system based on the outer membrane of engineered E. coli DH5α according to claim 2, characterized in that: In step (1), the centrifugation temperature of the centrifugal collection of E. coli DH5ɑ bacterial cell precipitate is 4℃, the centrifugal force is 5000g, and the time is 10 min; the ultrasonic crushing power is 300w, and the time is 30 min; the centrifugation temperature of the broken bacterial liquid is 4℃, the centrifugal force is 2900g, and the time is 1h; the Tris buffer solution is added to the precipitate, and the high-speed centrifugation centrifugation temperature is 4℃, the centrifugal force is 20000g, and the time is 1h; the supernatant is centrifuged again at high speed, the centrifugation temperature is 4℃, the centrifugal force is 125000-150000g, and the time is 2h.

5. The brain-targeting drug delivery system based on the outer membrane of engineered E. coli DH5a according to claim 2, characterized in that: In step (1), the Tris buffer solution added to the precipitate is specifically: 7.5 times the volume of 0.1 M Tris buffer solution is added to the precipitate wet weight, and the Tris buffer solution contains 10 mM EDTA and 5 mg / ml sodium deoxycholate. 6.The brain-targeting drug delivery system based on the outer membrane of engineered Escherichia coli DH5α according to claim 2, characterized in that: In step (2), the high polymer material is polylactic acid-glycolic acid, the organic solvent is ethyl acetate; the drug is an imaging agent, the mass ratio of the imaging agent to the high polymer material is 0.02:1-0.2:1, the imaging agent dissolved in the oil phase is any one of DiR, IR780 and SPIO, and the imaging agent dissolved in the water phase is doxorubicin. 7.The brain-targeting drug delivery system based on the outer membrane of engineered E. coli DH5α according to claim 2, characterized in that: In step (2), the external water phase is 2.5% polyvinyl alcohol or vitamin polyethylene glycol succinate solution; the volatile water phase is 0.3% polyvinyl alcohol or vitamin polyethylene glycol succinate solution. 8.The brain-targeting drug delivery system based on the outer membrane of engineered Escherichia coli DH5α according to claim 2, characterized in that: In step (2), the centrifugation temperature is 4℃, the speed is 30000-35000rpm, and the time is 20 min. 9.The brain-targeting drug delivery system based on the outer membrane of engineered Escherichia coli DH5α according to claim 2, characterized in that: In step (3), the mass ratio of the E. coli DH5ɑ modified outer membrane to the final precipitated nano-carrier is 1:5, the ultrasonic time is 3 min, and the extrusion times is 11.

10. The brain-targeting drug delivery system based on the modified E. coli DH5ɑ outer membrane according to claim 1 is used for preparing a drug delivery system targeting brain endothelial cells.

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