Preparation methods and applications of bacteria for drug delivery carriers

Abstract

This invention relates to a method for preparing bacteria for drug delivery carriers and their uses. Specifically, this invention relates to a method for preparing bacteria for drug delivery carriers, comprising: culturing bacteria; collecting bacterial cells in the logarithmic growth phase and resuspending them to obtain a suspension; incubating the suspension in a water bath at 53°C to 57°C for 4 to 6 minutes, then immediately immersing it in liquid nitrogen for 45 to 75 minutes, repeating this process three times to obtain bacteria for drug delivery carriers, wherein the bacteria are *Escherichia coli*. This invention also relates to the use of the bacteria or compositions used as drug delivery carriers in the preparation of pharmaceuticals.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical biochemistry, specifically to a method for preparing and using bacteria as a drug delivery carrier. Background Technology

[0002] Cancer, as the leading cause of death among humans, has made its treatment a highly sought-after research topic. Due to the significant differences between the tumor extracellular matrix (ECM) and normal tissue, therapeutic drugs are difficult to deliver effectively to solid tumor cells, thus hindering the targeting of key pathways. The tumor vascular system and extracellular matrix synergistically regulate the transvascular and interstitial transport of nanoparticles, which is crucial for the successful delivery of nanomedicines to solid tumors.

[0003] In recent years, microbial engineering has opened new avenues for the development of drug delivery vectors, therapeutic agents, and diagnostic methods by leveraging the targeting and gene packaging capabilities of bacteria. Escherichia coli and some anaerobic bacteria, with their rod-shaped morphology and biosurface, can efficiently penetrate the mucus barrier and tumor stroma, thus accumulating in large quantities at tumor sites. Furthermore, bacteria, with their inherent immunostimulatory and immunogenic properties, can trigger immune responses against tumors, making them ideal vectors for enhancing and guiding anti-tumor immune responses.

[0004] However, most common bacteria are pathogenic, and their colonization, proliferation, and drug release in the body are difficult to precisely control, potentially leading to off-target effects and uncontrollable side effects. Administering live bacteria poses varying degrees of harm to the human body, severely limiting the further application of bacterial therapy.

[0005] To improve the safety of therapeutic bacteria, a common strategy is to delete major virulence genes to obtain attenuated strains. However, this method is complicated in terms of gene knockout, limiting its rapid application; moreover, even after gene knockout, virulence cannot be guaranteed. Any residual virulence in these engineered bacteria could pose a potential threat to immunocompromised patients.

[0006] Therefore, designing and developing a simple and safe drug delivery system is of great importance. Summary of the Invention

[0007] In view of this, the first aspect of the present invention provides a method for preparing bacteria for a drug delivery carrier, comprising: culturing bacteria, collecting bacterial cells in the logarithmic growth phase and resuspending them to obtain a suspension; bathing the suspension in a water bath at 53°C to 57°C for 4 to 6 minutes, and then immediately immersing it in liquid nitrogen for 45 to 75 minutes, repeating the process three times to obtain bacteria for a drug delivery carrier, wherein the bacteria are Escherichia coli.

[0008] In some implementations, the suspension is placed in a water bath at 54°C to 56°C.

[0009] In some implementations, the suspension bath time is 5 minutes.

[0010] In some implementations, the suspension is immersed in liquid nitrogen for 55 to 65 minutes.

[0011] In some implementations, the cells in the logarithmic growth phase are resuspended using a CaCl2 solution.

[0012] In some implementations, scanning electron microscopy reveals that the bacteria used as drug delivery carriers maintain their intact structure.

[0013] In some implementations, the bacteria used as drug delivery vectors are not capable of proliferation.

[0014] In some implementations, the bacteria used as drug delivery vectors are not cytotoxic.

[0015] In some implementations, the bacteria used as drug delivery carriers maintain catalase activity.

[0016] A second aspect of the invention provides the use of bacteria or compositions for drug delivery carriers in the preparation of said drug, wherein the bacteria for drug delivery carriers are prepared according to the method of the first aspect of the invention.

[0017] The composition includes bacteria for drug delivery carrier and a drug adsorbed on the surface of the bacteria for drug delivery carrier.

[0018] In some implementations, the drug is directly adsorbed onto the bacterial surface used as a drug delivery carrier or adsorbed onto the bacterial surface used as a drug delivery carrier in the form of a nanocomposite.

[0019] In some embodiments, the nanocomposite includes a nanocarrier and a drug, wherein the nanocarrier includes at least one of polyethyleneimine (PEI) nanocarriers, poly-L-lysine (PLL) nanocarriers, polyamide-amine dendritic macromolecule (PAMAM) nanocarriers, poly(N,N-dimethylaminoethyl methacrylate) (PDMAEMA) nanocarriers, spermine nanocarriers, cationic polyurethane nanocarriers, chitosan nanocarriers, and cationic lipid nanocarriers.

[0020] In the method of this invention, by selecting specific water bath and liquid nitrogen treatment parameters and the number of repetitions for *Escherichia coli*, the harmfulness of the bacteria is significantly reduced, rendering it unable to proliferate and completely eliminating potential bacterial toxicity, thereby greatly improving the safety and controllability of bacterial treatment. Simultaneously, this treatment method preserves the basic morphology of the bacteria and most of its protein activity, allowing it to retain the structure and chemotaxis of viable bacteria. Furthermore, this treatment method is simple to implement, easy to operate, and suitable for large-scale application. Attached Figure Description

[0021] Figure 1 This is a flowchart illustrating the preparation of bacteria for drug delivery vectors according to the present invention.

[0022] Figure 2 The image shows the morphological characterization results of Escherichia coli in Example 2. Image a is a scanning electron microscope (SEM) image of the original Escherichia coli; image b is a scanning electron microscope (SEM) image of the Escherichia coli used as the drug delivery carrier.

[0023] Figure 3 The diagram shows the proliferation results of Escherichia coli in Example 3. In this diagram, a represents the growth curve of the original Escherichia coli; b represents the proliferation results of Escherichia coli under different treatments.

[0024] Figure 4 The graph shows the results of the catalase-catalyzed reaction of Escherichia coli under different treatments. In the graph, a represents the reaction results of the original E. coli; b represents the reaction results of E. coli inactivated at 100℃; c represents the reaction results of the 55*3 group of E. coli; d represents the reaction results of the 60*2 group of E. coli; e represents the reaction results of the 60*3 group of E. coli; f represents the reaction results of the 70*1 group of E. coli; g represents the reaction results of the 70*2 group of E. coli; and h represents the reaction results of the 70*3 group of E. coli.

[0025] Figure 5 This is a graph showing the safety analysis of RAW264.7 cells using *E. coli* (active bacteria) as the original strain and *E. coli* (shocked bacteria) as the drug delivery carrier. In the graph, 'a' represents a bacterial colony count of 0, 1×10⁻⁶. 5 2×10 5 3×10 5 4×10 5 6×10 5 8×10 5 10×10 5 12×10 5 Experimental results at CFU / mL; b represents bacterial colony count of 40 × 10⁻⁶. 5 60×10 5 80×10 5 100×10 5 120×105 160×10 5 180×10 5 200×10 5 Experimental results at CFU / mL.

[0026] Figure 6 This figure shows the targeting validation results of *E. coli* used as a drug delivery carrier. In the figure, a represents the experimental results of *E. coli* that does not express PD-1 protein; b represents the experimental results of *E. coli* that expresses PD-1 protein.

[0027] Figure 7 This is a graph showing the results of an E. coli loading experiment used as a drug delivery carrier. Detailed Implementation

[0028] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood in the technical field to which this invention pertains. The following definitions are supplementary to those definitions in the art and relate to this invention, but are not extrapolated to any relevant or unrelated situation, such as any conventionally used patent or application. While any methods and materials similar to or equivalent to those described herein may be used in practical testing of the invention, the materials and methods described herein are preferred. Therefore, the terminology used herein is intended to describe specific embodiments only and is not intended to limit the invention.

[0029] In this document, the terms "one or more" and "at least one" are used interchangeably.

[0030] In this document, the terms "one or more" and "at least one" are used interchangeably.

[0031] The terms “including,” “comprising,” or “having”, when used before a step or element, indicate the addition of a further step or element, which is optional and not excluded.

[0032] As mentioned above, the present invention aims to address the shortcomings of existing technologies by providing a convenient and safe drug delivery system. Based on this, the present invention provides a method for preparing bacteria for drug delivery carriers.

[0033] In this study, the bacteria used as drug delivery carriers were subjected to liquid nitrogen treatment (LNT) to a state of shock, and are therefore referred to as "shocked bacteria." In this study, the term "shock state" refers to the physiological stress state that bacteria enter upon exposure to liquid nitrogen. In the shock state, bacteria no longer possess the ability to proliferate, but retain the activity of their expressed proteins.

[0034] The term "drug delivery carrier" refers to a substance that carries drugs through physical encapsulation, chemical bonding, or adsorption, controls their release kinetics (such as sustained release or stimulus-response release), and overcomes physiological barriers (such as cell membranes and blood-brain barriers) to achieve precise delivery.

[0035] In a specific implementation, the method of the present invention includes culturing bacteria and collecting bacterial cells in the logarithmic growth phase.

[0036] By way of example, bacteria can be cultured in a culture medium containing an antibiotic corresponding to the resistance carried by the bacteria. The culture conditions for the bacteria can be those known in the art, for example, in the case of Escherichia coli, the culture conditions can be 37°C and 200 rpm.

[0037] The term "logarithmic growth phase" refers to the period after the lag phase in which the number of bacterial cells increases geometrically, i.e., by a factor of 2. 0 →2 1 →2 2 →···2 n The growth period can be measured using methods well-known in the art (e.g., measuring OD). 600 Confirm the culture status of the bacteria.

[0038] In some embodiments, the method of the present invention can culture bacteria and determine the logarithmic growth phase of the bacteria by plotting growth curves.

[0039] Exemplarily, the method for collecting bacterial cells in the logarithmic growth phase is achieved by centrifuging and washing the bacterial culture. The centrifugation conditions for the bacterial culture can be those known in the art, as long as they successfully precipitate the bacterial cells. For example, in the case of *Escherichia coli*, centrifugation conditions can be 6000 rpm for 5 minutes. In some specific embodiments, the centrifuged bacterial cells can be washed with a buffer or culture medium known in the art, for example, with a pH 7.4 phosphate buffer solution. In some specific embodiments, the same centrifugation conditions can be used after washing to obtain bacterial cells in the logarithmic growth phase.

[0040] In specific embodiments, the method of the present invention includes resuspending bacterial cells in the logarithmic growth phase to obtain a suspension. Exemplarily, suitable media can be used to resuspend the bacterial cells in the logarithmic growth phase. In some specific embodiments, a CaCl2 (0.1M) solution is used to resuspend the bacterial cells in the logarithmic growth phase.

[0041] In a specific implementation, the method of the present invention includes subjecting the suspension to a water bath.

[0042] For example, the temperature of the water bath is 53°C to 57°C, such as 53°C, 54°C, 55°C, 56°C or 57°C.

[0043] As demonstrated in the examples below, the inventors were surprised to find that even after three subsequent liquid nitrogen freeze-thaw treatments, E. coli could still proliferate when the water bath temperature was 37°C and 50°C; and that even a slight temperature adjustment, such as raising the water bath temperature from 50°C to 55°C, could affect the proliferation of E. coli; this discovery would have been difficult to anticipate before the experiments and explorations of this invention were carried out.

[0044] For example, the duration of a single water bath is 4 to 6 minutes. In some specific implementations, the water bath duration is 4 minutes, 4.5 minutes, 5 minutes, 5.5 minutes, or 6 minutes.

[0045] Unlike conventional short-time heat excitation, the water bath time in this invention is set to be relatively long. Surprisingly, the long-term high-temperature treatment did not destroy the activity of proteins in E. coli. On the contrary, as demonstrated in the examples below, E. coli that were water bathed at 55°C for 5 minutes and subjected to three freeze-thaw cycles still maintained the activity of catalase-catalyzed proteins.

[0046] For example, the suspension after water bath is immersed in liquid nitrogen and frozen for 45 to 75 minutes, such as 45 minutes, 46 minutes, 47 minutes, 48 ​​minutes, 49 minutes, 50 minutes, 51 minutes, 52 minutes, 53 minutes, 54 minutes, 55 minutes, 56 minutes, 57 minutes, 58 minutes, 59 minutes, 60 minutes, 61 minutes, 62 minutes, 63 minutes, 64 minutes, 65 minutes, 66 minutes, 67 minutes, 68 minutes, 69 minutes, 70 minutes, 71 minutes, 72 minutes, 73 minutes, 74 minutes, or 75 minutes.

[0047] In the specific implementation plan, the water bath and liquid nitrogen treatment were repeated three times.

[0048] In the specific implementation plan, there are no additional processing steps between the water bath and the liquid nitrogen treatment.

[0049] In the specific implementation plan, there are no additional processing steps between the three repetitions.

[0050] Additional processing steps include, but are not limited to, centrifugation, resuspending, and incubation at room temperature for more than 1 minute.

[0051] In this paper, scanning electron microscopy revealed that the bacteria obtained by the method of the present invention for drug delivery maintained an intact structure. Therefore, the prepared bacteria still possess the penetration characteristics and immune properties derived from the structure and chemotaxis of live bacteria, and remain suitable for use as drug delivery carriers.

[0052] In this study, the bacteria used as drug delivery vectors are not capable of proliferation. In some embodiments, the bacteria used as drug delivery vectors are not cytotoxic. Therefore, the prepared bacterial delivery vectors have high biosafety, which is beneficial for the further application of bacterial therapy.

[0053] In this study, the bacteria used as drug delivery carriers maintained catalase activity. This demonstrates that the method of the present invention can preserve the activity of expressed proteins, thereby maintaining the specific biological functions of the bacteria.

[0054] In some implementations, the bacteria used as drug delivery vectors can express proteins that are targets.

[0055] In some specific implementation schemes, the target protein may be a cancer target protein.

[0056] In some more specific implementations, the target protein may be the PD-1 protein.

[0057] The present invention also provides the use of bacteria or compositions for drug delivery carriers in the preparation of drugs, wherein the bacteria for drug delivery carriers are prepared according to the method of the present invention.

[0058] In some embodiments, the composition includes bacteria for a drug delivery carrier and a drug adsorbed on the surface of the bacteria for the drug delivery carrier.

[0059] In some embodiments, the drug is directly adsorbed onto the surface of the bacteria used as a drug delivery carrier. In other embodiments, the drug is adsorbed onto the surface of the bacteria in the form of a nanocomposite.

[0060] In some implementations, the nanocomposite includes a nanocarrier and a drug.

[0061] In some embodiments, the nanocarrier includes at least one of polyethyleneimine (PEI) nanocarriers, poly-L-lysine (PLL) nanocarriers, polyamide-amine dendritic macromolecule (PAMAM) nanocarriers, poly(N,N-dimethylaminoethyl methacrylate) (PDMAEMA) nanocarriers, spermine nanocarriers, cationic polyurethane nanocarriers, chitosan nanocarriers, and cationic lipid nanocarriers.

[0062] In some specific implementations, the nanocarrier is a cationic lipid nanocarrier.

[0063] In some implementations, the drug is a positively charged drug.

[0064] In some implementations, the drug is a non-electrified drug.

[0065] In some specific implementation schemes, the drug is at least one of the drugs used to treat tumors, inflammatory bowel disease, and obesity. For example, the drug is carboplatin.

[0066] The following describes preferred embodiments of the present invention, but the present invention is not limited to these preferred embodiments. It should be noted that any modifications and improvements made by those skilled in the art based on this inventive concept are within the scope of protection of the present invention. All reagents used, unless otherwise specified, are commercially available conventional products.

[0067] Example 1: Preparation of Escherichia coli for drug delivery carrier

[0068] The process for preparing the *E. coli* used as a drug delivery carrier in this embodiment is as follows: Figure 1 As shown, the general steps are as follows:

[0069] (1) Escherichia coli BL21 was cultured at 37°C in LB medium containing ampicillin (Amp) resistance;

[0070] (2) Collect Escherichia coli in the logarithmic growth phase, centrifuge at 6000 rpm for 5 minutes to obtain bacterial cells; wash the bacterial cells with an equal volume of pH 7.4 phosphate buffer solution in a clean bench, continue to centrifuge at 6000 rpm for 5 minutes, and repeat the washing three times; discard the supernatant, resuspend the precipitate with an equal volume of 0.1 mol / L CaCl2 solution (sterilized), collect and dispense into 1 mL centrifuge tubes;

[0071] (3) After suspending the centrifuge tube containing the bacteria, insert it into a 55°C water bath for 5 minutes, and then quickly immerse it in a liquid nitrogen tank for 60 minutes.

[0072] (4) Repeat (3) three times to obtain E. coli for drug delivery carrier.

[0073] Example 2: Morphological characterization of *Escherichia coli* used as a drug delivery carrier

[0074] After 2 hours of vacuum freeze-drying, the Escherichia coli prepared as a drug delivery carrier according to the method of Example 1 and the untreated original Escherichia coli were observed using a scanning electron microscope (Guoyi Quantum SEM5000X).

[0075] Experimental results are as follows Figure 2 As shown, it can be seen that the original Escherichia coli ( Figure 2 a) and Escherichia coli used as drug delivery carriers Figure 2 The basic morphology of b) is consistent, which explains why E. coli used as a drug delivery carrier maintains its complete cell structure.

[0076] Example 3: Proliferation of Escherichia coli

[0077] 3.1 Proliferation of primitive Escherichia coli

[0078] method:

[0079] (1) The original Escherichia coli was cultured in the same way as in Example 1. 200 μL of samples were taken at 0, 1, 2, 4, 6, 8, 12, 14, 16, 18, 20, 22, 30, 38, 42, 44, 46, and 48 hours of culture. The OD value of the samples was measured at 600 nm using an ELISA reader (BioTek Synergy H1M, USA) with uninoculated LB medium as a blank control.

[0080] (2) With OD 600 Plot the growth curve of E. coli with the growth value as the ordinate and the growth time as the abscissa.

[0081] result:

[0082] The growth curve of primitive E. coli is as follows Figure 3 As shown in Figure a, it can be seen that the logarithmic growth phase of E. coli is from 6 to 18 hours.

[0083] 3.2 Proliferation of Escherichia coli used as a drug delivery carrier

[0084] method:

[0085] (1) Escherichia coli for drug delivery carrier was prepared according to the method of Example 1, except that the temperature of the water bath was adjusted from 55°C in Example 1 to 37°C, 42°C, 50°C, 60°C and 70°C respectively, thus setting a total of 6 temperature groups of 37°C, 42°C, 50°C, 55°C, 60°C and 70°C, and the number of times of liquid nitrogen freeze-thaw at each temperature was set to 1 time, 2 times and 3 times respectively, for a total of 18 experimental groups, which were recorded as 37*1, 37*2, ​​37*3, 42*1, 42*2, 42*3, 50*1, 50*2, 50*3, 55*1, 55*2, 55*3, 60*1, 60*2, 60*3, 70*1, 70*2, 70*3;

[0086] (2) Take 100 μL of each group of Escherichia coli and culture it in the same way as in Example 1. After 18 hours of culture, take 200 μL of the sample and measure its OD value at 600 nm using an ELISA reader (BioTek Synergy H1M, USA).

[0087] result:

[0088] The proliferation results of E. coli used as drug delivery carriers in each group are as follows: Figure 3As shown in Figure b, it can be seen that after a 55°C water bath for 5 minutes and liquid nitrogen treatment for 1 hour, *E. coli* still proliferates rapidly after one or two repetitions, but after three repetitions, *E. coli* stops proliferating. When the water bath temperature rises to 60°C, after two or three repetitions of water bath and liquid nitrogen treatment, the proliferation of *E. coli* is affected, and *E. coli* stops proliferating. When the water bath temperature rises to 70°C, after one, two, or three repetitions of water bath and liquid nitrogen treatment, the proliferation of *E. coli* is affected, and *E. coli* stops proliferating.

[0089] Example 4: Catalase-catalyzed reaction of Escherichia coli

[0090] method:

[0091] (1) Take 1 mL of the original Escherichia coli, Escherichia coli inactivated at 100℃, and Escherichia coli with bacterial suspensions of 55*3, 60*2, 60*3, 70*1, 70*2 and 70*3 respectively and add them to the plate;

[0092] (2) Add 2 mL of 3% hydrogen peroxide solution to each group of bacterial culture. If bubbles are produced within 5 minutes, the group is considered positive; if no bubbles are produced, the group is considered negative.

[0093] result:

[0094] The reaction results of the original *E. coli*, *E. coli* inactivated at 100℃, and *E. coli* of 55*3, 60*2, 60*3, 70*1, 70*2, and 70*3 are as follows: Figure 4 As shown in Figures a to h, surprisingly, only the 55*3 bacterial culture produced a similar amount of bubbles as the original *E. coli*, while the *E. coli* inactivated at 100°C produced no bubbles. This suggests that the *E. coli* prepared by the method of this invention retained the activity of catalase-catalyzed proteins. The bacterial cultures treated at other temperatures and with liquid nitrogen produced significantly fewer bubbles, indicating very low catalyzed protein activity. Therefore, this study found that *E. coli* can be treated to a state suitable for use as a drug delivery carrier only when subjected to a water bath at 55°C and repeated freeze-thaw cycles with liquid nitrogen three times.

[0095] Example 5: In vitro safety validation of Escherichia coli used as a drug delivery carrier

[0096] method:

[0097] (1) Spread well-grown RAW264.7 cells at a density of 6 × 10⁶ cells per well. 3 100 μL of DMEM culture medium containing 5% fetal bovine serum was seeded into each well of a 96-well plate.

[0098] (2) After 24 hours of cell culture, the cell growth density was approximately 80%. The supernatant was discarded by centrifugation. The original *E. coli* and the *E. coli* prepared as a drug delivery carrier according to this invention (group "55*3" in Example 3) were cultured at 0 and 1 × 10⁻⁶ ppm, respectively. 5 2×10 5 3×10 5 4×10 5 6×10 5 8×10 5 10×10 5 12×10 5 , and 40×10 5 60×10 5 80×10 5 100×10 5 120×10 5 160×10 5 180×10 5 200×10 5 The bacterial colony count of CFU / mL was obtained by resuspending the bacterial culture in DEME without serum, and adding 100 μL of each concentration to three replicates in a 96-well plate.

[0099] (3) After incubating in a cell culture incubator for 24 hours, centrifuge and discard the supernatant. Add 90 μL of DMEM medium + 10 μL of CCK-8 solution to each well and incubate at 37°C for 30 minutes.

[0100] (4) Using a microplate reader (BioTek Synergy H1M, USA), the DMEM medium without inoculated cells was used as a blank control, and its OD value at 450 nm was measured. 450 value.

[0101] result:

[0102] The safety results of the original *E. coli* and the *E. coli* prepared in this invention for drug delivery in RAW264.7 cells are as follows: Figure 5 As shown (bacterial colony count is 0, 1×10⁻⁶), 5 2×10 5 3×10 5 4×10 5 6×10 5 8×10 5 10×10 5 12×10 5 The results at CFU / mL are as follows Figure 5 As shown in Figure a; the bacterial colony count is 40 × 10⁻⁶. 5 60×10 5 80×10 5 100×105 120×10 5 160×10 5 180×10 5 200×10 5 The results at CFU / mL are as follows Figure 5 As shown in Figure b), it can be seen that the Escherichia coli prepared by the present invention for drug delivery carriers did not show significant changes in cell activity under various concentration treatments, indicating that the Escherichia coli prepared by the present invention for drug delivery carriers will not cause serious cytotoxicity.

[0103] Example 6: Targeting validation of E. coli used as a drug delivery carrier

[0104] method:

[0105] (1) E. coli expressing PD-1 protein was obtained by gene modification. Ordinary E. coli BL21 was used as a control. E. coli for drug delivery vector was prepared by the treatment method "55*3" in Example 3, and PD-1LNT-E. coli and LNT-E. coli were obtained respectively.

[0106] (2) Transfer 4T1 cells to confocal dishes at an appropriate number of cells, culture overnight for 24 hours, then discard the culture medium, wash three times with PBS, cover the bottom of the cell dish with working solution of Hoechst 33342 dye, stain for 15 minutes, and wash three times with PBS.

[0107] (3) Stain LNT-E. coli and PD-1 LNT-E. coli with PI dye for 20 min, and wash three times with PBS;

[0108] (4) Add LNT-E. coli containing PI fluorescence and PD-1 LNT-E. coli to a confocal dish and incubate them together at 37°C for 3 h.

[0109] (5) After 3 hours, wash the confocal dish three times with PBS to remove unbound E. coli;

[0110] (6) LNT-E. coli and PD-1 LNT-E. coli were observed using a confocal microscope.

[0111] result:

[0112] The targeting validation results of LNT-E. coli and PD-1 LNT-E. coli are as follows: Figure 6As shown (a is the result of LNT-E. coli, b is the result of PD-1 LNT-E. coli), Hoechst 33342 dye can stain the cell nucleus blue, and PI dye can stain E. coli red. Figure 6 As can be seen, the red and blue regions in LNT-E. coli expressing PD-1 protein overlap, indicating that the Escherichia coli used as a drug delivery carrier of the present invention can target the cell surface after expressing PD-1 protein, indicating that the Escherichia coli used as a drug delivery carrier of the present invention retains the targeting activity of the expressed protein.

[0113] Example 7: Loading experiment of Escherichia coli for drug delivery carrier

[0114] method:

[0115] (1) Thoroughly mix Escherichia coli (the Escherichia coli prepared by the treatment method "55*3" in Example 3 for drug delivery carrier) with cationic lipid nanocarrier;

[0116] (2) Collect the bacterial cells by centrifugation at 6000 rpm for 5 minutes. The precipitate was washed three times with PBS to remove free cationic lipid nanocarriers.

[0117] (3) The potentials of cationic lipid nanocarriers, shock bacteria and shock bacteria bound to cationic lipid nanocarriers were detected respectively.

[0118] result:

[0119] Experimental results are as follows Figure 7 As shown, the bacteria without cationic lipid nanocarriers have a negative potential. After the cationic lipid nanocarriers bind to the bacterial surface through electrostatic interaction, the bacterial surface potential flips to positive, indicating that the cationic lipid nanocarriers are successfully adsorbed onto the shocked bacteria. This demonstrates that the prepared *E. coli* strain used as a drug delivery carrier can successfully adsorb cationic lipid nanocarriers.

[0120] In summary, the *Escherichia coli* (LNT-E. coli) used as a drug delivery carrier in this invention no longer has the ability to proliferate, does not cause serious cytotoxicity, and can maintain its intact structure and protein activity, and can adsorb nanocomposites including nanocarriers and drugs.

[0121] This document uses specific embodiments to illustrate the principles and implementation methods of the present invention. The descriptions of the embodiments above are merely for the purpose of helping to understand the method and central idea of ​​the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from its principles, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. A method for preparing bacteria for drug delivery carriers, characterized in that, include: Bacteria were cultured, and the cells in the logarithmic growth phase were collected and resuspended in CaCl2 solution to obtain a suspension. The suspension was incubated in a water bath at 55°C for 5 minutes, then immediately immersed in liquid nitrogen for 60 minutes. This process was repeated three times to obtain the bacteria used as a drug delivery carrier. The bacteria in question is Escherichia coli.

2. Use of the preparation method of claim 1 in the preparation of bacteria for drug delivery carriers.

3. The use as described in claim 2, characterized in that, The drug is directly adsorbed onto the bacterial surface of the drug delivery carrier or adsorbed onto the bacterial surface of the drug delivery carrier in the form of a nanocomposite. The nanocomposite is composed of a nanocarrier and the drug, wherein the nanocarrier is at least one of polyethyleneimine (PEI) nanocarrier, poly-L-lysine (PLL) nanocarrier, polyamide-amine dendritic macromolecule (PAMAM) nanocarrier, poly(N,N-dimethylaminoethyl methacrylate) (PDMAEMA) nanocarrier, spermine nanocarrier, cationic polyurethane nanocarrier, chitosan nanocarrier, and cationic lipid nanocarrier.

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