CN116606791A

CN116606791A - Drug-loaded minicells displaying endocytosis-promoting targeting membrane peptide and preparation method and application thereof

Info

Publication number: CN116606791A
Application number: CN202310439565.9A
Authority: CN
Inventors: 马毅; 朱冠舒; 王菊芳
Original assignee: South China University of Technology SCUT
Current assignee: South China University of Technology SCUT
Priority date: 2023-04-23
Filing date: 2023-04-23
Publication date: 2023-08-18

Abstract

The invention discloses a drug-loaded minicell for displaying an endocytic targeting membrane peptide, a preparation method and application thereof, wherein the surface of the drug-loaded minicell expresses a tumor cell targeting membrane peptide, and the amino acid sequence of the tumor cell targeting membrane peptide is shown as SEQ ID No. 2. The invention transfers the pBV220-arac-LOP recombinant plasmid into the engineering bacteria of the escherichia coli Nissle1917 delta minCD, and carries out protein expression to prepare a miniells carrier with the surface displaying endocytosis-promoting targeting membrane peptide, and then carries out drug incubation to prepare the drug Minicells. The drug-loaded minicells displaying the endocytic peptide can be produced in a large scale by a simple biological fermentation mode, are low in cost, can be effectively taken up by tumor cells, and enhance the anti-tumor effect of the drug.

Description

Drug-loaded minicells displaying endocytosis-promoting targeting membrane peptide and preparation method and application thereof

Technical Field

The invention belongs to the field of biological medicine, and in particular relates to a medicine-carrying minicell for displaying endocytic rational membrane peptide, and a preparation method and application thereof.

Background

Along with the popularization and younger onset of colorectal cancer, the research of colorectal cancer is deepened continuously, and the treatment of colorectal cancer is developed from the initial single operation treatment to the current endoscopic treatment, and the comprehensive means such as operation, radiotherapy, chemotherapy, targeted treatment and the like are integrated. However, since colorectal cancer lacks obvious early symptoms and a simple early diagnosis method, the early diagnosis rate of colorectal cancer in China is not high at present, 80% of patients have middle and late stages in the diagnosis, so that the subsequent treatment and survival rate are affected, and huge economic burden is caused to society and families. The targeting therapy is a novel therapeutic means for colorectal cancer, has unique advantages due to the accuracy of the targeting therapy in anticancer, particularly provides a novel method for treating advanced colorectal cancer, and can effectively prolong the survival time of patients. Thus, more molecules with unique effects in colorectal cancer are found, which can provide more ideas for colorectal cancer treatment. Recently, the incidence of cancer is increasing, and patients are in a trend of younger. The traditional treatment mode has low efficiency and large side effect, and can not meet the treatment requirement. Thus, there is an urgent need to develop new anticancer drugs. Bacteria have been promising for tumor therapy due to their unique ability. The lack of specificity and pertinence results in limited application and clinical research due to the large toxic and side effects of most bacteria themselves. Bacteria may be considered as programmable "robotic factories" specific for tumors, which possess unique capabilities that make them well suited as ideal anticancer agents. Recently, the mechanism of action of bacteria on tumor cells and antitumor action have been studied. Bacteria exhibit intrinsic antitumor activity because they express chemotactic receptors which direct chemotaxis of molecular signals in the tumor microenvironment. They are also fitted with flagella, which aids in tissue penetration. They can migrate and accumulate away from the vasculature. They may also be engineered to sense and respond to tumor microenvironments, thereby generating an innate and adaptive anti-tumor immune response. However, the antitumor effect of intratumoral bacteria is generally weak, and different bacteria and therapeutic strategies have been developed to enhance the antitumor effect. Although a plurality of novel nano-drug carriers are developed aiming at tumor characteristics at present, the problems of complex preparation steps, high cost, difficulty in realizing large-scale commercial production and the like remain key challenges for clinical transformation in the future. Therefore, the safe, effective and promising bionic material needs to be developed by using multidisciplinary technology, the treatment efficiency of tumor is improved, and the cost is reduced.

The research of bacteria as delivery vectors can be traced to the 90 s of the 20 th century, and the development of biotechnology has led to considerable success in the research of applications of bacteria as vectors in cancers, viral infections, diabetes and the like, but many challenges remain in thoroughly eradicating tumors in the face of complex tumor microenvironments: if living bacteria are used for treating tumors, there is a possibility that mutant strains may exist, normal organs of patients may face serious infections, and the patients may not be directly applied to organisms due to toxicity problems, and in addition, individual differences in treating tumors by using living bacteria are large and the repeatability is poor.

To take full advantage of the biological delivery system, such as bacteria, it must be deeply modified. In addition to living bacteria, a range of bacterial derivatives are also used as biological carriers for delivering antigens, drugs and the like, such as bacterial outer membrane vesicles, bacterial minicells, bacterial protoplast vesicles, bacterial ghosts and the like. Minicells are formed primarily by controlling bacterial division genes and inhibiting the polar sites of cell division. Systems that regulate cell division include the nuclear-like occlusion system and the Min system. The bacterial Min system consists of three genes, minC, minD and MinE. MinC or MinD gene mutations result in frequent isolation in the very vicinity of cells, rather than in intermediate cells, followed by the formation of small spherical minicells lacking chromosomal DNA or long filament-like cells containing chromosomal DNA. Similar to the parent bacterial cell, minicells also contain peptidoglycans, ribosomes, proteins, RNAs, and plasmids, but lack chromosomal DNA. Thus, minicells cannot grow or divide, but still retain the activity of other cells, including ATP synthesis, mRNA translation, transcription, and plasmid DNA translation. Thus, knocking out MinCD or over-expressing MinE in the bacterial genome can induce abnormal cell division, producing a large number of minicells. Secondly, as a unique biological delivery carrier, the minicells gather all the advantages of the traditional bacterial delivery system, the minicells have the advantages of the largest quantity in nature, simple sources, high preparation efficiency, low development cost and easy genetic engineering transformation, and are a safe and easy-to-operate bacterial substitute. Furthermore, the small cells with proper size are stable and the protein expressed by the thallus is enriched in the small cells, which is obviously superior to other delivery systems. Therefore, the small cells can be modified to have the exercise capacity of actively targeting focus tissues and promote endocytic absorption, which is one of factors limiting popularization and application, and further research and improvement are needed.

Disclosure of Invention

Aiming at the problems existing in the prior art, one of the purposes of the invention is to provide a preparation method of drug-loaded minicells with surface display and endocytosis promotion, wherein the polynucleotide sequence for encoding the surface display and endocytosis promotion membrane peptide is shown as SEQ ID NO. 1, and the amino acid sequence of the display system is shown as SEQ ID NO. 2. The second purpose of the invention is the application of the drug-loaded minicells displaying endocytic membrane peptide prepared by the method in the aspect of personalized tumor treatment.

A drug-loaded minicell displaying endocytosis-promoting targeting membrane peptide expresses tumor cell targeting membrane peptide on the surface of the drug-loaded minicell, and the amino acid sequence of the tumor cell targeting membrane peptide is shown as SEQ ID No. 2.

Preferably, the drug-loaded minicells are obtained by separating and purifying the MinC and MinD genes and the MinE gene overexpression by knocking out the MinC and MinD genes from escherichia coli Nissle 1917.

Preferably, the tumor cell targeting membrane peptide is expressed by transforming a recombinant plasmid into escherichia coli, wherein the recombinant plasmid comprises a signal peptide Lpp, an outer membrane protein OmpA and a coding gene of the tumor cell targeting membrane peptide.

Preferably, the coding gene of the tumor cell targeting membrane peptide is shown as SEQ ID No. 1.

Preferably, the minicell-loaded drug comprises doxorubicin and a pharmaceutically acceptable carrier and/or adjuvant thereof.

The preparation method of the drug-loaded minicells comprises the following steps:

(1) Constructing escherichia coli Nissle1917 with MinC and MinD genes knocked out and MinE genes overexpressed and recombinant plasmid pBV220-arac-LOP containing Lpp-OmpA-p28 genes, transforming the recombinant plasmid into competent cells of escherichia coli (EcN delta minCD: minE), and screening by taking kanamycin and ampicillin as resistance to obtain recombinant EcN engineering bacteria;

(2) Adding the recombinant EcN engineering bacteria obtained in the step (1) into a resistance culture medium, culturing for 4-5 hours, adding an inducer IPTG to induce the expression of tumor cell targeting membrane peptide p28, and finally collecting the small cells after the induction expression;

(3) And (3) incubating the precipitate obtained after the prepared minicells are centrifuged with a drug solution, and centrifugally washing to obtain drug-loaded minicells.

Preferably, the condition for the tumor cell-targeted membrane peptide p28 induction in the step (2) is OD ₆₀₀ 0.4 to 1.2.

Preferably, the conditions for incubation of the drug in step (3) are: the concentration of the drug solution is 50-250 mug/mL, the incubation time is 8-10 h, and the incubation temperature is 37+/-2 ℃; the washing adopts physiological saline.

Preferably, the condition for the tumor cell-targeted membrane peptide p28 induction in the step (2) is OD ₆₀₀ 0.8 to 1.2; the incubation concentration of the medicine in the step (3) is 200-250 mug/mL.

The application of the drug-loaded minicells in preparing drugs for preventing and treating tumor diseases.

Compared with the prior art, the invention has the following beneficial effects:

(1) The invention prepares the small cell carrier with endocytic membrane peptide displayed on the surface through a simple biological fermentation mode, has simple preparation operation and low cost, and the carrier has a complete bacterial functional structure, no biological activity, high safety and good biocompatibility.

(2) The small cell drug carrying system provided by the invention can also carry chemotherapeutic drugs, improves the anti-tumor effect of the drugs, and provides a brand new idea for personalized disease treatment.

(3) The drug-loaded Mini for displaying the endocytic peptide provided by the invention can be produced in a large scale by a simple biological fermentation mode, has low technical cost, can be effectively taken up by tumor cells, and enhances the anti-tumor effect of the drug.

Drawings

FIG. 1 is a plasmid map of pUC19-Lpp-OmpA-p 28.

FIG. 2 shows a EcN. DELTA. MinuCD:: minuE growth curve for Lpp-OmpA-p28 expression, and WT EcN. DELTA. MinuCD:: minuE.

FIG. 3 shows SDS-PAGE gel to verify the expression of Lpp-OmpA-p28, black arrow indicates protein band of Lpp-OmpA-p28, and WT is normal EcN. Delta. MinuCD:: minE.

FIG. 4 shows EcN. Delta. MinCD expressing Lpp-OmpA-p28 by minE immunofluorescence staining; a: ecN delta minCD:: minE, scale: 6.6 μm; b: expression of Lpp-OmpA-p28 protein EcN. Delta. MinuCD:: minE, scale bar: 6.6 μm.

FIG. 5 is a SEM, TEM, and fluorescence microscopy analysis of EcN. Delta. MinuCD:: minum forming minicells. A: bacteria produce a fluorescence map of minicells; b: fluorescence map of minicells after purification; c: SEM image of bacterial production minicells and minicell-to-bacteria separation; d: TEM images of individual minicells.

Fig. 6 is the effect of different drug incubation conditions on the encapsulation efficiency of minicells. A: drug concentration; b: incubation time; and C, in vitro drug release curve.

FIG. 7 is a fluorescent microscope view of Minicell _DOX 、Minicell ^p28 _DOX Different targeting experiments on normal colon cells NCM460 and colon cancer cells HT 29. A: HT29; b: NCM460.

FIG. 8 is a flow cytometer analysis Minicell _DOX 、Minicell ^p28 _DOX Different targeting experiments on normal colon cells NCM460 and colon cancer cells HT 29. ns p>0.05,*p<0.05,**p<0.01,***p<0.001。

FIG. 9 is a statistical analysis Minicell _DOX 、Minicell ^p28 _DOX MFI map internalized by cancer cells.

Detailed Description

In order that those skilled in the art will better understand the core technology of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments. It should be understood that the experimental methods used in the following examples are conventional methods unless otherwise specified.

1. Main experimental materials and sources:

(1) EcN DeltaMinCD:: minE is constructed on the basis of EcN/DeltatnaA:: T7RNAP, and EcN/DeltatnaA:: T7RNAP competent cells are constructed by referring to the paper "construction of a bacterial ghost efficient preparation system and preliminary application research thereof", normal colon cells NCM460 and colon cancer cells HT29 are both derived from the American standard biological collection center (ATCC), plasmid pKD46 is provided by Youbao organism, and the product number is VT1692.

(2) The main reagent comprises: yeast extract, tryptone was purchased from Oxoid;2.5% glutaraldehyde electron microscope special fixing solution is purchased from Feijing corporation; acrylamide, persulfate Amide (APS), tetramethyl ethylenediamine (TEMED), chloramphenicol, sucrose, BSA blocking solution, coomassie brilliant blue R-250, available from the division of bioengineering (Shanghai); mouse monoclonal ANTI-cellM2 antibody, sodium Dodecyl Sulfate (SDS) purchased from Sigma;26616 protein Marker was purchased from thermosipher; goat anti-mouse IgG H&L (Alexa Fluor 488) is purchased from Abcam; physiological saline, PBS, L-arabinose were purchased from Shanghai Biotechnology Co., ltd; doxorubicin hydrochloride was purchased from calicheamicin biotechnology limited; kanamycin and ampicillin are purchased from Aladin; agar powder, naCl, concentrated sulfuric acid, concentrated hydrochloric acid, glacial acetic acid, isopropanol, trehalose, mannitol and absolute ethanol are purchased from Tianjin far chemical reagent company; DL 2000DNA Marker and DL 5000DNA Marker were purchased from Takara corporation; protein markers were purchased from sameira company; plasmid extraction kits and universal DNA purification recovery kits were purchased from the tiangen biochemical technology (beijing) limited company; fetal bovine serum was purchased from Lonsera;

DMEM medium, penicillin/Streptomycin, 0.25% Trypsin-EDTA available from Gibco; CCK-8 kit was purchased from Dojindo.

Example 1: preparation of recombinant EcN engineering bacterial strain EcN delta minCD:: minE

Firstly, synthesizing a fusion fragment of kanamycin and RBS by PCR, transferring a pKD46 plasmid into EcN/delta tnaA by using an electric shock method, preparing a competent cell of T7RNAP to obtain bacteria with recombinase, transferring the fusion fragment into a new competent cell by using the electric shock method to perform homologous recombination, knocking out MinC and MinD genes and overexpression of MinE genes, and screening by taking kanamycin as a resistance under the transformation conditions of 1.5KV, 200 omega and 25 mu F.

Example 2: construction of pBV 220-arac-Lpp-OmpA-p28 plasmid

Based on the sequences of plasmids pBV220-sGFP-Amp, pKD46 and pUC19-Lpp-OmpA-p28 (plasmid map see FIG. 1), the upstream and downstream primers were designed using the seamless cloning method, and the primer sequences were as follows:

TABLE 1 Membrane peptide genes Lpp-OmpA-p28, araC-ParaBAD and linearized pBV220-Amp PCR amplification primers

Preparing a PCR system, carrying out PCR amplification, wherein the PCR system and the program are as follows:

TABLE 2 PCR amplification System and reaction procedure

Taking 4 mu L of PCR amplified products for agarose gel electrophoresis analysis; purifying and recovering to obtain target gene

Lpp-OmpA-p28, araC-ParaBAD and linearized pBV220-Amp fragments;

the method of seamless cloning is adopted, the recovered PCR product is taken as a fragment and a carrier for linear amplification, and a PCR reaction system is as follows:

table 3 seamless cloning reaction System and reaction procedure

Transforming the seamless cloning product into E.coli DH5 alpha competence, and screening by taking ampicillin as resistance; the next day, selecting a monoclonal, performing colony PCR by using a specific primer pBV220-Amp-F/P28-R, and sequencing positive clones with correct band sizes; positive clone plasmid with correct sequencing is successfully constructed recombinant plasmid, which is named as pBV

220-arac-Lpp-OmpA-p28；

Example 3: effect of Lpp-OmpA-p28 expression on bacterial Activity

The recombinant EcN engineering bacteria strain obtained in example 1 was added to LB liquid medium containing 50. Mu.g/mL kanamycin and 100. Mu.g/mL ampicillin, and cultured overnight at 37℃and 220 rpm. The following day, the transfer is carried out according to the ratio of bacterial liquid of 1:100, ecN delta minCD:: minE (WT) is used as a control, and the sample is sampled once per hour and passes through OD ₆₀₀ The change in the values was used to estimate bacterial growth, as shown in FIG. 2, with the recombinant EcN engineered strain growing slower than the control.

In addition, after inoculation, samples were taken of EcN. Delta. MinCD:: minE/pBV 220-arac-Lpp-OmpA-p28, induced with L-arabinose at concentrations of 0.25mg/mL, 0.5mg/mL and 1mg/mL, respectively, for observation of Lpp-OmpA-p28 protein expression. Bacteria after protein expression were collected by centrifugation at 12000rpm for 2min, and the collected cells were completely suspended at a ratio of 500. Mu.LPBS per 5 OD. The thoroughly suspended thalli are placed in an ice box, bacteria are crushed for 5min by using an ultrasonic cell crusher, the mode is phi 2, the ultrasonic power is 70W,3s is on, and 3s is off. After the cell disruption, 80. Mu.L was aspirated, and 20. Mu.L of 5×loading Buffer was added thereto, and after complete mixing, the mixture was boiled at high temperature for 10min and centrifuged at 12000rpm for 2min. Firstly, preparing 15% of separating glue, after the plane is finished by using 1mL of isopropanol, carefully taking the absorbent paper to suck the absorbent paper, and then adding the prepared 5% of concentrated glue. The sample wells on both sides of the gel were first loaded with 10. Mu.L of 1 Xloading buffer to prevent the strip from being distorted, and then the treated samples were loaded into the sample wells in a predetermined order with 10. Mu.L of the treated samples, and 5. Mu.L of protein Marker was added to one side of the sample wells. Adding a proper amount of 1 XTris-Gly electrophoresis buffer solution into an electrophoresis tank, covering the electrophoresis tank, inserting a power line plug into a power jack (red to red and black to black) of an electrophoresis apparatus, turning on a switch, adjusting the voltage to 80V, running for 20-30 min to see a narrow band, then changing 120V to about 60-75 min, changing specific electrophoresis time according to the size of a target strip to be imprinted, referencing the position of a protein Marker or the position of a bromophenol blue indication dye (the indication dye just runs out of a glass plate below, and changing the color of the electrophoresis tank into light blue), and ending electrophoresis. After electrophoresis, the glass plate is pried off, concentrated glue is cut off, the separated glue is put into a glue box, coomassie blue staining solution is added, the mixture is heated for 10 seconds by a microwave oven, and then the mixture is placed into a small shaking table for staining for 15-20 minutes. Discarding the staining solution, washing off residual staining solution with tap water, adding decolorizing solution into a staining box, decolorizing in a small shaking table until blue background disappears, and finally photographing the decolorized gel under a scanner to preserve the picture. Meanwhile, after the gel is run normally, the gel is carefully taken out, the redundant gel part is cut off, the cut gel is carefully placed on an NC film, bubbles between the gel and the film are removed, the sandwich of a film transferring instrument is clamped, and then the film is transferred for 60min at 90V. After turning well, the membranes were blocked in 5% nonfat milk powder for 1h, washed 3 times with 1XTBST for 10min each, and mouse anti-Flag (HRP) was added in 1:3000 columns, incubated overnight at 4 ℃ and then membranes were washed 3 times with TBST for 10min each. And taking out 1mL of each of the solution A and the solution B from the Super Signal West Pico chemiluminescent substrate kit, and uniformly mixing. And (3) opening the exposure instrument, precooling to-21 ℃, then placing the NC film on a sample tray of the exposure instrument, carefully dripping the AB liquid which is uniformly mixed on the size of the target strip, closing the sample tray, and performing exposure, photographing and storage through software.

As shown in FIG. 3, ecN. DELTA. MinCD expressing Lpp-OmpA-p28 was induced by culturing in the presence of a target protein band after induction, but the protein expression level did not increase with increasing arabinose concentration, compared with normal EcN. DELTA. MinCD:: minE, so that induction was performed at a concentration of 0.5 mg/mL. Since the Lpp-OmpA display system needs to be displayed on a bacterial membrane, the expression process of a toxic protein for bacteria, and the overexpression of the Lpp-OmpA display system can have a great influence on the cell viability. In combination with FIG. 1, it is presumed that the increase in resistance to culture conditions and the expression of the smaller Lpp-OmpA-p28 protein increased the growth burden of EcN. Delta. MinuCD:: minE, resulting in slow growth initiation and decreased cell viability.

Example 4: immunofluorescence observation of distribution of Lpp-OmpA-p28 in cells

In order to clearly see the distribution of the Lpp-OmpA-p28 protein in bacteria, we used the Flag tag on the protein to perform antigen-antibody reaction to trace its localization. Bacterial liquid was collected at 4000rpm for 5min, the supernatant was discarded, and the culture was washed three times with 1 XPBS to remove the remaining LB medium. After blocking with BSA blocking solution for 1h at room temperature, bacteria were collected and washed 3 times with 1 XPBS. Adding mouse monoclonal ANTI-cell diluted in the ratio of 1:3000The M2 antibody was incubated overnight at 4 ℃. The bacterial sediment after the primary antibody is incubated is continued to be treated with goat anti-mouse IgG H&L(Alexa />488 Incubation for 2h at room temperature (dilution ratio 1:500). Fluorescence signals from the bacterial surface were captured using a confocal laser scanning microscope.

It can be seen from FIG. 4A that no fluorescence was observed in the control group, while more green fluorescence was observed in FIG. 4B, and the fluorescence was uniformly distributed on the surface of the cells, and since the fluorescence was not in the same focal plane, some of the cells might not be collected, indicating that Lpp-OmpA-p28 protein was successfully displayed on the surface of the cells.

Example 5: fluorescence microscope, scanning electron microscope and transmission electron microscope for observing minicell morphology

After pUC19-GFP plasmid is introduced into EcN delta minCD engineering bacteria, the bacteria can spontaneously express green fluorescent protein, and then the minicells before and after purification are observed by a fluorescent microscope.

And collecting bacterial precipitate, adding a proper amount of 2.5% glutaraldehyde electron microscope special fixing solution for fixing overnight at 4 ℃, centrifuging, washing to remove the fixing solution, washing with ultrapure water, re-suspending on a cell climbing plate, and embedding in a filter paper plate. Gradient dehydration treatment is carried out in 70%, 85% and 95% ethanol, repeated three times, and finally soaked in 100% ethanol for standby. Sequentially placing the treated samples into a carbon dioxide critical point dryer for drying, and taking out the samples for metal spraying treatment after the procedure is finished; finally, the morphology of the minicells was observed under a field emission scanning electron microscope.

The sampling step of the transmission electron microscope is similar to the sampling step of the scanning electron microscope, the thallus is resuspended by 500 mu L of ultrapure water, 10 mu L of resuspended thallus is sucked and dripped on a common carbon film copper net, the mixture is stood for 5min, and then the bacterial liquid on the copper net is carefully sucked by a 10 mu L pipetting gun. 10. Mu.L of 3% tungsten phosphate dye was dropped onto the copper mesh, and the solution was allowed to stand for 3 minutes, followed by careful blotting with a gun head. 10. Mu.L of ultrapure water was dropped onto the copper mesh, and carefully sucked away to wash out excess staining solution. Standing and air-drying, and observing the sample by using a transmission electron microscope.

As shown in FIG. 5A, an engineering bacterium containing an expressed green fluorescent protein was cultured to OD ₆₀₀ Sampling at=1.1 to take a sample of the process of bacterial production minicells, removal of minicells of the parental bacteria after purification is clearly visible in fig. 5B. From the results of the scanning electron microscope, it can be seen (FIG. 5C) that the bacteria are small cells that produce a sphere at the pole. As can be seen from fig. 5D, the purified minicells were visualized under transmission electron microscopy as having a membrane structure.

Example 6: the optimal drug loading conditions of minicells were explored.

1. Each group was aspirated 1mL of purified minicells, and the prepared serial concentration DOX solution was added, blown and mixed well, incubated at 37 ℃ for 60min at 220rpm, at which time the solution was observed to turn red, centrifuged at 10000xg for 5min, and the supernatant removed. Washing with physiological saline for 5min 3 times. Finally, the drug content in the minicells was determined and the encapsulation efficiency calculated. As shown in FIG. 6A, the encapsulation efficiency of minicells increases with increasing drug concentration over a range of concentrations, so the drug incubation temperature of the present invention is preferably 250 μg/mL of drug solution.

2. Each group was aspirated 1mL of purified minicells in a 1.5mL centrifuge tube, added with the same concentration of DOX solution, mixed well, incubated for 10h at a temperature of 37 ℃ at 220rpm, sampled every two hours, centrifuged for 5min at 10000xg, and the supernatant removed. Washing with physiological saline for 5min 3 times. Finally, the drug content in the minicells was determined and the encapsulation efficiency calculated. As shown in fig. 6B, the encapsulation efficiency of minicells increases with increasing incubation time over a range of concentrations, so the drug incubation time of the present invention is preferably overnight incubation.

Example 7: and evaluating the in-vitro drug release capacity of the drug-loaded minicells.

Drug-loaded minicells were dissolved in release medium (BSG buffer) and placed in a constant temperature shaking table at 37℃and 220 rpm. Taking 1mL of release external liquid at 0, 2, 4, 6, 8, 10, 12, 14 and 16h, firstly carrying out ultrasonic crushing on the external liquid, measuring the absorbance of the release medium at 480nm, and calculating the encapsulation efficiency of the medicine. As shown in fig. 6C, the minicells were not prone to leakage after drug loading, likely due to metabolic inactivation caused by the minicells' lack of bacterial genomes themselves. The drug carrying capacity of minicells is largely derived from the fact that minicells retain bacterial membranes and thus contain large amounts of transport proteins.

Example 8: in vitro targeting experiments of drug-loaded minicells.

NCM460 and HT29 cells in the logarithmic phase were seeded in 12-well plates at a cell seeding density of 2X10 ⁵ Culturing the cells/holes in a constant temperature incubator with 5% CO2 at 37 ℃ for overnight; the next day the medium was discarded and 500. Mu.L of DMEM medium containing 2% FBS was added again, 500. Mu.LMinicells _DOX 、Minicells ^p28 _DOX (same concentration of DOX), cells were treated. Placing the mixture in a constant temperature incubator with 5% CO2 at 37 ℃ for continuous incubation and culture for 2 hours; the old broth was then discarded, washed 3 times with 1×pbs to wash out unabsorbed DOX from the system, and observed under a fluorescent microscope, and representative field shots were taken. Meanwhile, after 2h incubation with the drug, the old medium was discarded, washed 3 times with PBS, digested with pancreatin, and after completion, the pellet was collected by centrifugation, washed 3 more times with PBS, and resuspended in PBS for fluorescence intensity analysis by flow cytometer.

As shown in FIGS. 7A and B and FIG. 8, minicells ^p28 _DOX Fluorescence intensity ratio in cancer cellsIn normal cells, and is stronger than in non-minicels _DOX Strong in cancer cells. This suggests that cancer cells internalize more Minicells displaying the endocytosis-promoting targeting membrane peptide p28, validating Minicells ^p28 _DOX Is endocytosis promoting and targeting. HT29; NCM460.

The above embodiments are only preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments, and it should be apparent to those skilled in the art that any modifications, equivalent substitutions, improvements, etc. within the spirit and principle of the present invention are included in the scope of the present invention.

Claims

1. The drug-loaded minicells displaying endocytosis-promoting targeting membrane peptides are characterized in that the drug-loaded minicells express tumor cell targeting membrane peptides on the surfaces, and the amino acid sequences of the tumor cell targeting membrane peptides are shown as SEQ ID No. 2.

2. The drug-loaded minicells according to claim 1, wherein said drug-loaded minicells are obtained by isolation and purification by overexpression of the MinC and MinD genes and the MinE gene from E.coli Nissle1917 knockout.

3. The drug-loaded minicell of claim 2, wherein the tumor cell targeting membrane peptide is expressed by transformation of a recombinant plasmid into escherichia coli, the recombinant plasmid comprising a signal peptide Lpp, an outer membrane protein OmpA, and a gene encoding the tumor cell targeting membrane peptide.

4. The drug-loaded minicell of claim 3, wherein the coding gene of the tumor cell targeting membrane peptide is shown in SEQ ID No. 1.

5. The drug-loaded minicell of claim 1 or 2 or 3 or 4, wherein the minicell-loaded drug comprises doxorubicin and a pharmaceutically acceptable carrier and/or adjuvant thereof.

6. The method for producing a drug-loaded minicell according to any one of claims 1 to 5, comprising the steps of:

(1) Constructing escherichia coli Nissle1917 with MinC and MinD genes knocked out and MinE genes overexpressed and recombinant plasmid pBV220-arac-LOP containing Lpp-OmpA-p28 genes, transforming the recombinant plasmid into competent cells of the escherichia coli, and screening by taking kanamycin and ampicillin as resistance to obtain recombinant EcN engineering bacteria;

7. The method of claim 6, wherein the tumor cell-targeted membrane peptide p28 induction in step (2) is performed under the condition of OD ₆₀₀ 0.4 to 1.2.

8. The method of claim 7, wherein the incubation conditions for the drug in step (3) are: the concentration of the drug solution is 50-250 mug/mL, the incubation time is 8-10 h, and the incubation temperature is 37+/-2 ℃; the washing adopts physiological saline.

9. The method of claim 8, wherein the tumor cell-targeted membrane peptide p28 induction in step (2) is performed under the condition of OD ₆₀₀ 0.8 to 1.2; the incubation concentration of the medicine in the step (3) is 200-250 mug/mL.

10. Use of the drug-loaded minicells of any one of claims 1-5 in the manufacture of a medicament for the prevention and treatment of neoplastic disease.