Drug-loaded bacterial shadow displaying acid-triggered rational membrane peptide and preparation method and application thereof

Abstract

The present invention relates to the field of biomedicine, and discloses a drug-loaded bacterial ghost displaying an acid-triggered rational membrane peptide, and a preparation method and application thereof. The surface of the drug-loaded bacterial ghost expresses an acid-triggered rational membrane peptide that is sensitive to an acidic environment; the acid-triggered rational membrane peptide is transformed into an Escherichia coli strain by a plasmid vector pLysS for expression, and its amino acid sequence is shown in SEQ ID No: 2. The drug-loaded bacterial ghost displaying an acid-triggered rational membrane peptide provided by the present invention can be mass-produced in a simple manner, and can be effectively taken up by tumor cells to enhance the anti-tumor effect of the drug. The drug-loaded bacterial ghost is used in the preparation of drugs for the prevention and treatment of diseases characterized by a slightly acidic environment.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a drug-loaded bacterial shadow displaying acid-triggered rational membrane peptides, and a preparation method and application thereof. Background Art

[0002] Cancer is one of the most serious diseases that threaten human health. Traditional drug treatments lack specificity and inevitably produce toxic side effects on normal tissues while killing tumor cells, which greatly reduces the effectiveness of clinical treatment. At the same time, the complex physiological barriers and adverse immune environment in tumor tissues have increased the difficulty of traditional drug treatment, and new treatment methods are urgently needed. Although many new nano-drug carriers have been developed based on tumor characteristics, their complex preparation steps, high costs, and difficulty in large-scale commercial production are still key challenges for their future clinical transformation. Therefore, it is necessary to use multidisciplinary technologies to develop safe, effective, and promising bionic materials to improve the efficiency of tumor treatment and reduce costs.

[0003] As the most abundant organism in nature, bacteria have therefore come into the sight of researchers. The study of bacteria as delivery carriers can be traced back to the 1990s, when researchers used live Salmonella as antigen carriers for mucosal vaccines [Chatfield SN, et al. Vaccine. 1989, 7 (6): 495-498]. In the 21st century, the development of biotechnology has also driven the application of bacteria as carriers in cancer, viral infection, diabetes, etc., especially in the field of tumor treatment [Hosseinidoust Z., et al. Adv Drug Deliv Rev. 2016, 106 (Pt A): 27-44].

[0004] Studies have shown that bacteria escape from the blood circulation into tumor tissues through active or passive mechanisms. The active mechanism originates from bacterial motility organs such as pili, which allow bacteria to penetrate into the tissues. In addition, chemokines secreted in the tumor area have a chemotactic effect on bacteria [Kasinskas RW, et al. Biotechnol Bioeng. 2006, 94(4): 710-721]. There are also interaction dynamics between bacteria and hosts [Zhou S., et al. Nat Rev Cancer. 2018, 18(12): 727-743], which can guide free bacteria to tumor tissues. The passive mechanism is mainly manifested in that bacteria are captured by the chaotic vascular system in the tumor area, and the tumor tissue microenvironment is more conducive to bacterial colonization, growth and reproduction. Importantly, bacteria can also directly destroy tumor tissue by directly inducing oncolysis [Wang Y., et al. J Cancer. 2019, 10(19):4442-4454], destroying blood vessels to induce tumor thrombosis [Yi X., et al. Sci Adv. 2020, 6 (33):eaba3546], inflammation-induced cytotoxicity, and competition for nutrients [Chen F., et al. Biomaterials. 2019, 214:119226]. Therefore, bacteria themselves can be used as a biotherapeutic agent to treat tumors, such as Suresh et al. Caulobacter crescent As a monotherapy strategy for anti-tumor treatment, bacteria induce inflammatory responses in addition to inducing the secretion of necrosis factor TNF-α [Bhatnagar PK, et al. Cancer Biol Ther. 2006, 5 (5): 485-491]. Although bacteria have achieved considerable success in tumor treatment, there are still many challenges to completely eradicate tumors in the face of the complex tumor microenvironment: if live bacteria are used to treat tumors, there may be a risk of mutant strains, and the patient's normal organs may also face serious infections. There is also the possibility that they cannot be directly applied to the body due to toxicity issues, such as Salmonella Typhimurium [Toso JF, et al. J Clin Oncol. 2002, 20 (1): 142-152] and others including Clostridium novyi [Dang LH, et al. Proc Natl Acad Sci US A. 2001, 98 (26):15155-15160] and Listeria monocytogenes[Freitag NE, et al. Infect Immun. 1993, 61 (6): 2537-2544] anaerobic or facultative strains need to be attenuated to improve safety when used for tumor treatment. In addition, the use of live bacteria to treat tumors has large individual differences and poor reproducibility.

[0005] To fully utilize the advantages of bacteria as a biological delivery system, it is necessary to deeply transform them. In addition to live bacteria, a series of bacterial derivatives are also used as biological carriers to deliver antigens, drugs and other substances, such as bacterial outer membrane vesicles, bacterial minicells, bacterial protoplast vesicles and bacterial ghosts. Bacterial ghosts are a microbial-based biological delivery system, first reported by Austrian scholar Lubitz in the 1980s [Witte A., et al. European journal of biochemistry. 1989, 180 (2): 393-398]. Generally speaking, the empty shell of Gram-negative bacteria formed by the regulated expression of bacteriophage φX174 lytic protein E is a bacterial ghost [Szostak MP, et al. Journal of Biotechnology. 1996, 44 (1-3): 161-170]. Bacterial ghosts are an ideal carrier for personalized tumor treatment. Their main advantages are high safety, retaining the original cell morphology of natural live bacteria without genetic material, and no risk of horizontal gene transfer. Studies have shown that bacterial ghosts from different bacterial sources have no cytotoxic or genotoxic effects on human cells [Kudela P., et al. J Biotechnol. 2011, 153 (3-4): 167-175]. Secondly, as a unique biological delivery carrier, bacterial ghosts gather all the advantages of traditional bacterial delivery systems. They are the most abundant in nature, have simple sources, high preparation efficiency, low development cost, and are easy to genetically engineer. They are a safe and easy-to-operate bacterial substitute. Another is the storage stability of bacterial ghost products. Freeze-dried bacterial ghosts remain stable at room temperature for several years without being affected. Vaccines made from bacterial ghosts can be processed and stored without refrigeration, which is obviously better than other delivery systems. However, bacterial ghosts also lack the mobility of live bacteria to actively target lesion tissues, which is also one of the factors limiting their promotion and application, and further research and improvement are needed. Summary of the invention

[0006] In view of the problems existing in the prior art, one of the objects of the present invention is to provide a method for preparing drug-loaded bacterial ghosts displaying acid-triggered rational membrane peptide (ATRAM) on the surface, wherein the nucleotide sequence encoding the surface-displayed ATRAM is shown in SEQ ID NO:1, and the amino acid sequence of the display system is shown in SEQ ID NO:2.

[0007] The second purpose of the present invention is to use the drug-loaded bacterial shadows displaying ATRAM prepared by the above method in personalized tumor treatment and diseases with slightly acidic environment as pathological characteristics.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] A drug-loaded bacterial ghost displaying an acid-triggered rational membrane peptide, wherein the surface of the drug-loaded bacterial ghost expresses an acid-triggered rational membrane peptide that is sensitive to an acidic environment.

[0010] Preferably, the acid-triggered rational membrane peptide is transformed into an E. coli strain by a plasmid vector pLysS for expression, and its amino acid sequence is shown in SEQ ID No: 2. The gene sequence of the expression vector includes the gene sequences of the signal peptide Lpp, the outer membrane protein OmpA and the acid-triggered rational membrane peptide ATRAM.

[0011] Preferably, the bacterial shadow is Escherichia coli Nissle 1917 ( Escherichia coli Nissle 1917, EcN) bacterial shadow.

[0012] Preferably, the bacterial ghost is formed by the expression regulation of bacteriophage α3 lytic gene E.

[0013] Preferably, the bacterial ghost is prepared by expressing the Lpp-OmpA-ATRAM gene and then inducing the expression of bacteriophage α3 lytic protein E.

[0014] The preparation method of the drug-loaded bacterial shadow comprises the following steps:

[0015] (1) constructing a recombinant plasmid pET29a-α3-E containing the phage α3 lytic gene E and a recombinant plasmid pLysS-LOA containing the Lpp-OmpA-ATRAM gene, transforming the above recombinant plasmids into competent cells of the engineering bacteria, and screening with kanamycin and chloramphenicol as resistance to obtain recombinant EcN engineering bacteria;

[0016] (2) adding the recombinant EcN engineered bacteria obtained in step (1) to a resistant culture medium for cultivation, then adding an inducer IPTG to induce the expression of lytic protein E, and finally collecting the bacterial shadows after induced expression for freeze-drying;

[0017] (3) Incubate the prepared bacterial ghost lyophilized powder with the drug solution, centrifuge and wash, and obtain drug-loaded bacterial ghosts.

[0018] Preferably, the lysis protein E in step (2) induces OD 600 The freeze-drying protective agent is mannitol.

[0019] Preferably, the drug incubation concentration in step (3) is 2.5-7.5 mg / mL, the incubation time is 15-60 min, and the incubation temperature is 4°C-55°C.

[0020] Preferably, the lysis protein E in step (2) induces OD 600 It is 2.0~2.1.

[0021] Preferably, the drug incubation concentration in step (3) is 5.0-7.5 mg / mL; the incubation time is 60 min; and the incubation temperature is 37°C.

[0022] Preferably, the pH of the Tris-HCl buffer used for drug incubation in step (3) is 7.4-8.0.

[0023] Preferably, the drug loaded by the bacterial ghosts includes doxorubicin (DOX) and a pharmaceutically acceptable carrier and / or adjuvant thereof.

[0024] The drug-loaded bacterial shadows are used in the preparation of anti-tumor drugs and in the prevention and treatment of diseases (such as tumors) with a slightly acidic environment as a pathological feature.

[0025] The invention transfers two recombinant plasmids, pLysS-LOA and pET29a-α3-E, into EcN engineered bacteria by electroporation, and performs protein expression to prepare bacterial ghost carriers that display acid-triggered rational membrane peptides on the surface. At the same time, DOX is used as a drug model to explore its drug loading efficiency, and human liver cancer HepG2 cells are used as a cell model to explore its cytotoxicity and function. Experiments have shown that the drug-loaded bacterial ghosts that display acid-triggered rational membrane peptides provided by the present invention can be mass-produced in a simple manner, and can be effectively taken up by tumor cells, enhancing the anti-tumor effect of the drug.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] (1) The present invention uses a DNA recombination method to express ATRAM on a bacterial shadow vector, so that it has the function of actively targeting the acidic microenvironment of tumors. The method is simple to operate and low in cost. In addition, the vector retains a complete bacterial capsid structure, has no biological activity, is highly safe, has good biocompatibility, and has broad application potential.

[0028] (2) The bacterial shadow drug delivery system provided by the present invention can also carry chemotherapy drugs to enhance the anti-tumor effect of the drugs, which can provide a new idea for personalized disease treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 EcN / Δ for expressing Lpp-OmpA-ATRAM tnA::T7 RNAP growth curve, WT is EcN / Δ tnA ::T7 RNAP;

[0030] Figure 2 The SDS-PAGE gel verifies the expression of Lpp-OmpA-ATRAM. The black arrow points to the protein band of Lpp-OmpA-ATRAM. WT is the normal EcN / Δ tnA ::T7 RNAP;

[0031] Figure 3 EcN / Δ for expressing Lpp-OmpA-ATRAM tnA ::T7 RNAP immunofluorescence staining; A: EcN / Δ tnA ::T7 RNAP, scale bar: 6.6 μm; B: expression of Lpp-OmpA-ATRAM protein EcN / Δ tnA ::T7 RNAP, scale bar: 6.6 μm; C: expression of Lpp-OmpA-ATRAM protein EcN / Δ tnA ::T7 RNAP, scale bar: 30.9 μm;

[0032] Figure 4 EcN / Δ at different induction starting points tnA :: Lysis curve assay of T7 RNAP / (pLysS-LOA + pET29a-α3-E);

[0033] Figure 5 EcN / Δ for expressing Lpp-OmpA-ATRAM tnA :: SEM and TEM analysis of T7 RNAP. A: TEM image of complete bacterial morphology; B: TEM image of bacterial shadow morphology; C: SEM image of complete bacterial morphology; D: SEM image of bacterial shadow morphology; E: SEM image of bacterial shadow within a larger field of view, with the white arrow pointing to the transmembrane pore of the bacteria;

[0034] Figure 6 The effects of different drug incubation conditions on drug loading and encapsulation efficiency of bacterial ghosts. A: drug concentration; B: incubation temperature; C: Tris-HCl buffer pH; D: incubation time;

[0035] Figure 7 is the in vitro drug release curve of drug-loaded bacterial shadow;

[0036] Figure 8 Toxicity experiments of Free DOX, BG-DOX and A-BG-DOX on HepG2 cells under different pH culture medium conditions; A: pH 6.5; B: pH 7.4.

[0037] Fig. 9 This is a diagram for evaluating the blood compatibility of drug-loaded bacterial shadows. DETAILED DESCRIPTION

[0038] In order to enable those skilled in the art to better understand the core technology of the present invention, the present invention will be further described below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the experimental methods used in the following implementation cases are all conventional methods unless otherwise specified.

[0039] 1. Main experimental materials and sources:

[0040] (1) EcN and human liver cancer HepG2 cells were both obtained from the ATCC Biological Standard Resource Center in the United States. The pLysS-OmpA-ATRAM plasmid and pET29a-E-α3 plasmid (Chinese patent CN 114736273A has been published) were synthesized by Genewise.

[0041] (2) Main reagents: Yeast extract and trypsin were purchased from Oxoid; 2.5% glutaraldehyde electron microscopy fixative was purchased from Feijing Company; acrylamide, ammonium persulfate (APS), tetramethylethylenediamine (TEMED), chloramphenicol, sucrose, BSA blocking solution, and Coomassie Brilliant Blue R-250 were purchased from Sangon Biotechnology (Shanghai) Co., Ltd.; mouse monoclonal ANTI-FLAG® M2 antibody and sodium dodecyl sulfate (SDS) were purchased from Sigma; 26616 protein marker was purchased from Thermofisher; goat anti-mouse IgG H&L (Alexa Fluor 488) was purchased from Abcam; Tris-HCl buffer and PBS were purchased from Shanghai Baisai Biotechnology Co., Ltd.; doxorubicin hydrochloride was purchased from Dalian Meilun Biotechnology Co., Ltd.; kanamycin and β-isopropylthiogalactoside (IPTG) were purchased from Aladdin; agar powder, NaCl, concentrated sulfuric acid, concentrated hydrochloric acid, glacial acetic acid, isopropanol, and anhydrous ethanol were purchased from Tianjin Zhiyuan Chemical Reagent Company; fetal bovine serum was purchased from Lonsera; DMEM medium, Penicillin / Streptomycin, and 0.25% Trypsin-EDTA were purchased from Gibco; and CCK-8 kit was purchased from Glpbio.

[0042] Example 1: Preparation of recombinant EcN engineered bacterial strains

[0043] Preparation of EcN / Δ tnA ::T7 RNAP competent cells, using electroporation to transform pLysS-OmpA-ATRAM plasmid and pET29a-E-α3 plasmid into EcN / Δ tnA:: In T7 RNAP competent cells, the transformation conditions were: 1.5 KV, resistance 200Ω, capacitance 25 μF, and kanamycin and chloramphenicol were used as resistance for screening to obtain the recombinant strain.

[0044] Example 2: Effect of Lpp-OmpA-ATRAM expression on bacterial activity

[0045] The recombinant EcN engineered bacterial strain obtained in Example 1 was added to LB liquid culture medium containing 50 μg / mL kanamycin and 25 μg / mL chloramphenicol, and cultured overnight at 37°C and 220 rpm. The next day, the bacterial liquid was transferred at a ratio of 1:100 to obtain EcN / Δ tnA ::T7 RNAP was used as a control and samples were collected every hour to analyze the OD 600 The change in the value is used to estimate the bacterial growth, such as Figure 1 As shown, the growth of the recombinant EcN engineered bacterial strain is slower than that of the control group.

[0046] In addition, starting from 3 h after inoculation, EcN / Δ tnA::T7 RNAP / pLysS-LOA +pET29a-α3-E was sampled, and the sample was continuously taken for 8 h to observe the expression of Lpp-OmpA-ATRAM protein. The bacteria after protein expression were collected by centrifugation at 12000 rpm for 2 min, and the collected bacteria were completely suspended according to the ratio of adding 500μL PBS for every 5 OD. The thoroughly suspended bacteria were placed in an ice box, and the bacteria were broken for 5 min using an ultrasonic cell disruptor, the mode was φ2, the ultrasonic power was 70 W, 3 s on, and 3 s off. After the bacteria were broken, 80 μL was drawn, and 20 μL 5×LoadingBuffer was added. After mixing completely, it was boiled at high temperature for 10 min and centrifuged at 12000rpm for 2 min. First, prepare 12% separation gel, hydraulically flatten the surface with 1 mL of isopropanol, carefully dry it with absorbent paper, and then add the prepared 5% concentrated gel. First, add 10 μL of 1× Loading buffer to the sample wells on both sides of the gel to prevent the bands from running crooked. Then add 10 μL of the processed samples to the sample wells in a predetermined order, and add 5 μL of protein marker to one side of the sample well. Add an appropriate amount of 1× Tris-Gly electrophoresis buffer to the electrophoresis tank, cover the electrophoresis tank lid, and plug the power cord into the electrophoresis instrument power jack (red to red, black to black), turn on the switch, first adjust the voltage to 80 V and run for 20~30min, a narrow band can be seen, then change to 120 V, about 60~75 min, the specific electrophoresis time varies according to the size of the target band to be blotted, refer to the protein marker position or the position of the bromophenol blue indicator dye (the indicator dye just runs out of the lower glass plate, and the color of the electrophoresis tank turns light blue), and end the electrophoresis. After the electrophoresis is completed, pry open the glass plate, cut off the concentrated gel, put the separation gel into the gel box, add Coomassie blue staining solution, heat in a microwave for 10 s, and then place it on a small shaker for staining for 15~20min. Discard the staining solution, wash off the residual staining solution with tap water, add destaining solution to the staining box, place it on a small shaker for destaining until the blue background disappears, and finally place the destained gel under a scanner to take a picture and save the image.

[0047] like Figure 2 As shown, compared with the normal EcN / Δ tnA ::T7 RNAP compared to EcN / Δ expressing Lpp-OmpA-ATRAM tnA ::T7 RNAP cultured for 3 hours, small target protein bands began to appear, but the protein expression level did not change much over time. Since the Lpp-OmpA display system needs to be displayed on the bacterial membrane, it is a toxic protein expression process for bacteria. Overexpression of the Lpp-OmpA display system will have a significant impact on cell viability. Figure 1, it is speculated that the expression of the smaller Lpp-OmpA-ATRAM protein increased the EcN / Δ tnA ::The growth burden of T7 RNAP leads to a slow growth starting point, decreased bacterial viability, and early arrival of the plateau phase.

[0048] Example 2: Immunofluorescence observation of the distribution of Lpp-OmpA-ATRAM in bacteria

[0049] In order to clearly see the distribution of Lpp-OmpA-ATRAM protein in bacteria, we used the Flag tag on the protein to perform antigen-antibody reaction to trace its location. Collect the bacterial solution at 4000 rpm for 5 min, discard the supernatant, wash three times with 1×PBS, and remove the remaining LB medium. After blocking with BSA blocking solution at room temperature for 1 h, collect the bacteria and wash three times with 1×PBS. Add mouse monoclonal ANTI-FLAG®M2 antibody diluted at a ratio of 1:3000 and incubate overnight at 4°C. The bacterial pellet after incubation with the primary antibody was further incubated with goat anti-mouse IgG H&L (Alexa Fluor®488) at room temperature for 2 h (dilution ratio of 1:500). Use a laser confocal scanning microscope to capture the fluorescent signal on the bacterial surface.

[0050] from Figure 3 A shows that no fluorescence was observed in the control group, while Figure 3 In B, more green fluorescence is observed, and the fluorescence is evenly distributed on the surface of the bacteria. Because they are not in the same focal plane, the fluorescence of some bacteria may not be collected. When the field of view is enlarged ( Figure 3 C) In EcN / Δ expressing Lpp-OmpA-ATRAM protein tnA We can observe stronger green fluorescence signals in ::T7 RNAP, which fully demonstrates that the Lpp-OmpA-ATRAM protein is successfully displayed on the bacterial surface.

[0051] Example 3: Determination of the lysis curve of EcN expressing Lpp-OmpA-ATRAM

[0052] Pick EcN / Δ from the plate tnA ::T7 RNAP / pLysS-LOA + pET29a-α3-E monoclonal colonies were cultured in the corresponding LB liquid medium overnight and transferred to new medium the next day for further culture. 600 When the OD value reached about 0.8, 1.4, and 2.1, 1 mM IPTG was added to induce the expression of phage α3 lytic protein E. After that, the OD value of the bacterial solution was measured every 1 h. 600 Value, according to the bacterial solution OD 600 The degree of bacterial lysis was estimated by the decrease in the value.

[0053] like Figure 4 It can be seen that when the initial OD 600 When the OD value of the bacterial solution was about 0.85, IPTG was added to induce protein lysis. 600 It can drop to 0.31, and then OD 600 There was a slight increase to 0.35; when the bacteria were close to the plateau phase, the induction was carried out, and EcN / Δ tnA ::T7 RNAP from OD 600 The decrease was from 2.06 to 0.86, with a decrease greater than 1.0, which means that the lytic protein E used in the present invention can still fully exert its lytic effect on the bacteria in the presence of Lpp-OmpA-ATRAM protein; even when the bacterial growth state is close to the plateau phase, it is not affected. It can be seen that this system has great potential for the application of low-cost large-scale preparation of bacterial shadows. In summary, the preferred lytic protein of the present invention induces OD 600 is 2.0, and the induction time is 4 h.

[0054] Example 4: Observation of bacterial shadow morphology using scanning electron microscope and transmission electron microscope

[0055] Collect the bacterial precipitate, add an appropriate amount of 2.5% glutaraldehyde electron microscope fixative solution and fix it overnight at 4°C, then centrifuge and wash to remove the fixative, finally wash with ultrapure water and resuspend on the cell slide, and embed in the filter paper. Dehydrate in 70%, 85%, and 95% ethanol for gradient dehydration, repeat three times, and finally soak in 100% ethanol for standby use. Put the processed samples into the carbon dioxide critical point dryer in sequence for drying. After the program is completed, take out the samples for gold spraying; finally, observe the bacterial shadow morphology under a field emission scanning electron microscope.

[0056] The sampling steps for transmission electron microscopy are similar to those for scanning electron microscopy. Resuspend the bacteria with 500 μL ultrapure water, take 10 μL of the resuspended bacterial solution and drop it on the ordinary carbon film copper grid, let it stand for 5 minutes, and then use a 10 μL pipette to carefully remove the bacterial solution on the copper grid. Take 10 μL of 3% tungsten phosphate staining solution and drop it on the copper grid, let it stand for 3 minutes, and then carefully suck it away with the tip of the pipette. Take 10 μL of ultrapure water and drop it on the copper grid, carefully suck it away to wash away the excess staining solution. Let it stand and air dry, and observe the sample using a transmission electron microscope.

[0057] like Figure 5 The intact bacteria shown in A are darker in color and have a regular and smooth shape. Figure 5 In B, it is clearly visible that the bacteria shells are of different shades and have a high light transmittance. Figure 5 C and Figure 5D) Most of the transmembrane pores have only one and exist at both ends of the bacteria, while the rest of the structure remains intact. Some bacteria have depressions, which may be due to the osmotic pressure that causes the contents to flow out, turning into an "empty bag". This also means that the bacterial shadow has a large and complete cavity that can be used as a carrier for material transportation. Figure 5 As can be seen from E, most bacteria have transmembrane pores, while a few do not. This may be the result of mutant strains that are resistant to lysis.

[0058] Example 5: Exploration of the Optimal Drug Loading Conditions for Bacterial Shadows

[0059] 1. Accurately weigh 30 mg of bacterial ghost freeze-dried powder into a 1.5 mL centrifuge tube, add ultrapure water to dissolve, centrifuge at 4000 rpm for 10 min to collect the bacterial precipitate, and remove the supernatant. Add the prepared Tris-DOX solution of a series of concentrations to the bacterial precipitate, incubate at 37°C for 60 min, at which time the bacterial precipitate can be observed to turn red, centrifuge at 6000 rpm for 3 min, and remove the supernatant. Wash with Tris-HCl buffer (pH 8.0) 3 times, 5 min each time, and wash with pure water for the last time. Finally, determine the drug content in the bacterial ghost and calculate the drug loading and encapsulation efficiency. Figure 6 As shown in A, within a certain concentration range, the drug loading and encapsulation efficiency of bacterial shadows increase with the increase of drug concentration. Therefore, the drug incubation temperature of the present invention is preferably a 7.5 mg / mL drug solution.

[0060] 2. Accurately weigh 30 mg of bacterial shadow freeze-dried powder into a 1.5 mL centrifuge tube, add ultrapure water to dissolve, centrifuge at 4000 rpm for 10 min to collect bacterial precipitate, and remove the supernatant. Add Tris-DOX to the bacterial precipitate, incubate at different temperatures, centrifuge at 6000 rpm for 3 min, remove the supernatant, and treat the bacterial precipitate as before. Figure 6 As shown in Figure 2, when the bacterial shadows were incubated with the drug at 4, 16, and 25°C, there was no significant difference in the drug loading and encapsulation efficiency. However, when the temperature was raised to 37°C, the drug loading and encapsulation efficiency also increased. Even when the incubation temperature of the system reached 55°C ( p <0.05), the drug loading and encapsulation efficiency of bacterial shadows were the highest, which were 12.28% and 24.6%, respectively. This may be because the cell membrane of bacterial shadows is mainly composed of phospholipid bilayers, and high temperature greatly increases the fluidity of the membrane. The aromatic benzene ring of DOX makes it easier for DOX molecules to bind to the membrane, which may also cause the drug release rate to slow down. However, it should also be noted that excessively high temperatures may affect proteins such as antigens on the surface of bacterial shadows. At the same time, since 37°C is used for bacterial culture, the effect on the bacterial shadow itself is small under this temperature condition, so the drug incubation temperature of the present invention is preferably 37°C.

[0061] 3. Accurately weigh 30 mg of bacterial shadow freeze-dried powder into a 1.5 mL centrifuge tube, add ultrapure water to dissolve, centrifuge at 4000 rpm for 10 min to collect bacterial precipitate, and remove the supernatant. After adding Tris-HCl buffer containing different pH values ​​to the bacterial precipitate and incubating with the drug, centrifuge at 6000 rpm for 3 min, remove the supernatant, and treat the bacterial precipitate as before. Figure 6 As shown in Figure C, the drug loading and encapsulation efficiency of bacterial shadows in pH 8.0 Tris-HCl buffer environment are higher than those in pH 6.8 Tris-HCl buffer. It is speculated that the alkaline environment neutralizes the hydrochloric acid in doxorubicin hydrochloride, causing doxorubicin hydrochloride to become doxorubicin, with a decrease in water solubility and an increase in lipid solubility, making it easier to bind to the membrane. Low pH is not conducive to the binding of this drug to bacterial shadows. Within the research range, the drug loading and encapsulation efficiency of bacterial shadows are pH-dependent.

[0062] 4. Accurately weigh 30 mg of bacterial shadow freeze-dried powder into a 1.5 mL centrifuge tube, add ultrapure water to dissolve, centrifuge at 4000 rpm for 10 min to collect bacterial precipitate, and remove the supernatant. Add Tris-DOX solution and incubate the bacterial precipitate at 37°C for different time periods, centrifuge at 6000 rpm for 3 min, remove the supernatant, and treat the bacterial precipitate as before. Figure 6 As shown in Figure D, DOX can be loaded into bacterial ghosts after incubation in the system for 15 min. During the one-hour incubation time, the drug loading and encapsulation efficiency of bacterial ghosts increased with time in a time-dependent manner. The reason may be that the extension of time increases the chance of DOX combining with bacterial ghosts, but too long incubation time may also increase the instability of the system.

[0063] Example 6: Evaluation of in vitro drug release ability

[0064] The drug-loaded bacterial shadow (A-BG-DOX) with ATRAM displayed on the surface was dissolved in 1 mL of release medium (pH 5.5, pH 6.5, pH7.4 PBS buffer) and loaded into a dialysis bag. It was immersed in a 50 mL centrifuge tube containing 19 mL of release medium. It was placed in a constant temperature shaking shaker at 37°C and 150 rpm. 1 mL of release external liquid was taken at 0, 2, 4, 6, 12, 24, 48, and 72 h, and 1 mL of PBS buffer of the corresponding pH was added. The absorbance of the release medium was measured at 480 nm, and the cumulative release of the drug was calculated.

[0065] like Figure 7As shown in the figure, at pH 7.4, the cumulative drug release rate of A-BG-DOX within 2 h was 10.9%, and the cumulative drug release after 24 h remained below 18.9%. This is because the bacterial ghost, as a bacterial derivative, has a relatively stable structure under the physiological conditions of pH 7.4, which can slow down the diffusion of drugs in the carrier. The early drug release may be due to the rapid release of DOX adhering to the bacterial ghost and DOX existing in the bacterial ghost cavity. At pH 5.0, the cumulative drug release rate of 2 h was 21.9%, reaching 39.1% after 24 h, and 42.1% after 72 h, which is higher than the cumulative drug release rates of pH 6.5 and 7.4 in the same period. The increase in the cumulative drug release from pH 7.4 to 5.0 may be due to the increase in the solubility of DOX in water under slightly acidic conditions, or it may be that the acidic environment destroys the original stable structure of the bacterial ghost, resulting in increased drug release.

[0066] Example 7: In vitro cytotoxicity assay

[0067] HepG2 cells in the logarithmic growth phase were seeded in a 96-well plate with a cell seeding density of 5000 cells / well and cultured overnight in a 37°C, 5% CO2 constant temperature incubator. The next day, the culture medium was discarded and 100 μL of DMEM culture medium (pH 7.4 and pH 6.5) containing 2% FBS was added again. The cells were then treated with free drugs (Free DOX), drug-loaded bacterial ghosts without surface display of ATRAM (BG-DOX), and A-BG-DOX (drug doses of 0, 31.65, 62.5, 125, and 250 μg / mL). The cells were placed in a 37°C, 5% CO2 constant temperature incubator and cultured for 24 h. The culture medium was then discarded and washed with 1×PBS to remove bacterial shadows in the system and reduce the effect on absorbance. DMEM culture medium containing 10% CCK-8 was then added to each well. The wells with culture medium and CCK-8 were used as blank wells, and the wells with culture medium and CCK-8 were used as control wells. The cells were incubated in a 37°C, 5% CO2 constant temperature incubator. After the cells were colored, the absorbance at 450 nm was read with an enzyme reader and the cell survival rate was calculated.

[0068] like Figure 8As shown in the figure, under normal physiological conditions, Free DOX is more cytotoxic to HepG2 cells than BG-DOX and A-BG-DOX. In a slightly acidic environment, the survival rate of cells treated with free DOX (250 ng / mL) was 90.5%, while the survival rate of cells treated with A-BG-DOX decreased to 77.3%, and that of BG-DOX decreased to 87.5%. Similarly, the survival rate of cells treated with 31.25µg / mL A-BG-DOX was 90.3%, and the survival rates of cells treated with free DOX and BG-DOX were 98.9% and nearly 100%, respectively. At the same time, the study found that overall, the survival rate of HepG2 tumor cells at pH 6.5 was higher than that at pH 7.4. This is because chemotherapy drugs, especially weakly alkaline drugs such as doxorubicin, are easily protonated in the slightly acidic tumor environment. The drug's ability to penetrate the membrane is reduced, resulting in an inability to enter the cell and accumulation outside the cell, making it difficult to reach a concentration that kills tumor cells, resulting in physiological drug resistance. At the same time, the slightly acidic external environment may also promote tumor invasion and metastasis. Therefore, abnormal pH is one of the signs of malignant tumors.

[0069] Example 8: Evaluation of blood compatibility of drug-loaded bacterial ghosts.

[0070] Fresh periocular blood from C57 mice was collected in an anticoagulated blood vessel and centrifuged at 1000 rpm for 10 min to remove the supernatant. The cell pellet was then washed repeatedly with pre-cooled 1×PBS until the supernatant had no obvious hemoglobin color. Finally, pre-cooled 1×PBS was added to dilute the cell pellet to a final concentration of 4% and stored at 4°C. A-BG-DOX with a series of drug concentrations (20, 10, 5, 2.5, and 1.25 μg / mL) was added at a ratio of 1:1 (v / v), 1×PBS was used as a negative control, and 0.1% Triton X-100 was used as a positive control. The cells were incubated in a 37°C incubator for 1 h, centrifuged at 1000 rpm for 5 min, and the supernatant was collected. The absorbance of the supernatant at 540 nm was measured and calculated. Fig. 9 It shows that at the test concentration (10μg / mL), A-BG-DOX showed almost no hemolytic toxicity, with a hemolysis rate of less than 5%, while 0.1% Triton X-100 almost dissolved all red blood cells. This shows that the blood compatibility of drug-loaded bacterial ghosts is high and there is little damage to red blood cells after entering the blood.

[0071] The above-mentioned implementation cases are only preferred implementation methods of the present invention, but the implementation methods of the present invention are not limited to the above-mentioned implementation cases. Technical personnel in the relevant technical field should understand that any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A drug-loaded bacterial shadow displaying an acid-triggered rational membrane peptide, characterized in that: The surface of the drug-loaded bacterial ghost expresses an acid-triggered rational membrane peptide that is sensitive to an acidic environment; the acid-triggered rational membrane peptide is transformed into an Escherichia coli strain via a plasmid vector pLysS for expression, and its amino acid sequence is shown in SEQ ID No:

2.

2. The drug-loaded bacterial ghost according to claim 1, characterized in that: The bacterial shadow is the bacterial shadow of Escherichia coli Nissle 1917.

3. The drug-loaded bacterial ghost according to claim 1 or 2, characterized in that: The bacterial ghost is formed by the expression regulation of the phage α3 lytic gene E.

4. The method for preparing the drug-loaded bacterial ghost according to claim 1, 2 or 3, characterized in that: The following steps are involved: (1) constructing a recombinant plasmid pET29a-α3-E containing the phage α3 lytic gene E and a recombinant plasmid pLysS-LOA containing the Lpp-OmpA-ATRAM gene, transforming the above recombinant plasmids into Escherichia coli competent cells, and screening with kanamycin and chloramphenicol as resistance to obtain recombinant Escherichia coli engineered bacteria; (2) adding the recombinant Escherichia coli obtained in step (1) to a resistant culture medium for cultivation, then adding an inducer IPTG to induce the expression of lytic protein E, and finally collecting the bacterial shadows after induced expression for freeze-drying; (3) Incubate the prepared bacterial ghost lyophilized powder with the drug solution, centrifuge and wash, and obtain drug-loaded bacterial ghosts.

5. The method for preparing drug-loaded bacterial ghosts according to claim 4, characterized in that: The OD value of the bacterial solution during the induction of lysis protein E expression in step (2) is 600 is 0.8~2.1; the freeze-dried protective agent is mannitol; The incubation concentration of the drug solution in step (3) is 2.5-7.5 mg / mL, the incubation time is 15-60 min, and the incubation temperature is 4°C-55°C.

6. The method for preparing drug-loaded bacterial ghosts according to claim 5, characterized in that: The OD value of the bacterial solution during the induction of lysis protein E expression in step (2) is 600 2.0~2.1; The incubation concentration of the drug solution in step (3) is 5.0-7.5 mg / mL; the incubation time is 60 min; and the incubation temperature is 37°C; The solvent of the drug solution in step (3) is Tris-HCl buffer with a pH of 7.4 to 8.

0.

7. The preparation method according to claim 4, 5 or 6, characterized in that: The medicine comprises doxorubicin and a pharmaceutically acceptable carrier and / or adjuvant thereof.

8. The use of the drug-loaded bacterial ghost according to any one of claims 1 to 3, characterized in that: The drug-loaded bacterial shadow is used in the preparation of a drug for preventing and treating a malignant tumor disease with a slightly acidic environment as a pathological feature.

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