Escherichia coli drug-loaded preparation with photosensitive drug release function and preparation method of escherichia coli drug-loaded preparation

Abstract

The invention belongs to the technical field of biomedical materials, and provides a preparation method of a photosensitive drug release escherichia coli drug-loaded preparation, which comprises the following steps: incubating photosensitizer Ce6 nanoparticles and escherichia coli carrying 5-fluorouracil and prunella vulgaris extract to obtain the photosensitive drug release escherichia coli drug-loaded preparation, wherein the escherichia coli carrying the 5-fluorouracil and the selfheal extract is obtained by transferring the 5-fluorouracil and the selfheal extract into escherichia coli by adopting a high-voltage electroporation method; the photosensitizer Ce6 nano particles are prepared by dropwise adding tripolyphosphate into a Ce6 chitosan solution; the prunella vulgaris extract is obtained by extracting prunella vulgaris with water, concentrating an extracting solution and drying. According to the invention, the traditional anti-tumor drug 5-fluorouracil and the extract of the natural plant selfheal are matched to generate a synergistic effect in the anti-tumor aspect, and a certain reference value is provided for developing new anti-tumor drug compositions and preparations.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical materials, relates to drug-carrying bacterial agent technology, and specifically relates to a photosensitized drug-releasing Escherichia coli drug-carrying preparation and a preparation method thereof. Background Art

[0002] Cancer has become a major threat to human health. In recent years, researchers have often focused on new and reliable drug controlled release systems for anti-tumor purposes, aiming to protect drugs from rapid clearance in the blood, achieve targeted drug accumulation on tumors, enhance therapeutic effects, and reduce side effects. However, the current drug delivery system is not perfect in terms of drug loading and targeting. It is necessary to develop new tumor-targeted drug carriers to promote the targeted accumulation of drugs at the tumor site, promote the effective penetration of drugs in tumor tissues, and release drugs in response to stimulation signals.

[0003] Bacteria can drive flagella to convert chemical energy into mechanical energy to swim in the environment, and can sense different temperature, pH, oxygen and chemical inducer concentration gradients to swim directional [6] Therefore, researchers began to use the motility and tumor targeting ability of bacteria to prepare bacterial drug delivery systems.

[0004] Escherichia coli Nissle 1917 (EcN) is a facultative anaerobic probiotic found in humans and animals. It is non-toxic and is often used to prevent and treat intestinal diseases. Studies have shown that EcN has strong tumor targeting ability. EcN has limited tumor ablation ability, and the amount of exogenous protein expression in the body of genetically engineered bacteria is difficult to control, and there are safety issues with genetic modification.

[0005] In order to solve the above shortcomings, drug loading inside living bacteria can be considered to keep the bacteria active and tumor-targeting. Related to this, what kind of drugs can be loaded into bacteria to effectively play an anti-tumor role is a topic that needs to be studied urgently. Among traditional anti-tumor drugs, 5-fluorouracil is a commonly used drug. Whether 5-fluorouracil can be loaded into bacteria and play an anti-tumor role, and exploring what substances can produce better effects with 5-fluorouracil in bacteria, is also very important. Summary of the invention

[0006] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a method for preparing a photosensitive drug-releasing Escherichia coli drug-loaded preparation, which needs to overcome the shortcomings of insufficient anti-tumor effect of traditional bacterial anti-tumor technology and the lack of drug combinations that can produce synergistic anti-tumor effects with 5-fluorouracil. Thus, a drug-loaded preparation is provided that uses bacteria as tumor-targeting carriers and exerts a high-efficiency anti-tumor effect.

[0007] To this end, the present invention provides the following technical solutions:

[0008] A method for preparing a photosensitive drug-releasing Escherichia coli drug-loaded preparation, the preparation method comprising the following steps:

[0009] The photosensitizer Ce6 nanoparticles and Escherichia coli carrying 5-fluorouracil and Prunella vulgaris extract are incubated to obtain;

[0010] Wherein, the Escherichia coli carrying 5-fluorouracil and Prunella vulgaris extract is obtained by transferring 5-fluorouracil and Prunella vulgaris extract into Escherichia coli by high-voltage electroporation method;

[0011] The photosensitizer Ce6 nanoparticles are prepared by dropping tripolyphosphate into Ce6 chitosan solution;

[0012] The herba selfhei extract is obtained by extracting the herba selfhei with water, concentrating the extract and drying it.

[0013] Furthermore, the Escherichia coli is Escherichia coli Nissle 1917.

[0014] Furthermore, when performing high-voltage electroporation, 5-fluorouracil and Prunella vulgaris extract were dissolved in an electroporation buffer consisting of 0.5 M sorbitol, 0.5 M mannitol and 10% v / v glycerol.

[0015] Furthermore, when performing high-voltage electroporation, bacteria were harvested at mid-log phase and diluted to an OD of 600 The pH was 1.0, cooled in an ice bath for 5 minutes, and the bacterial suspension was transferred to an electroporation buffer containing 5-fluorouracil and Prunella vulgaris extract, and transferred to an electroporation cup with a gap of 0.2 cm, maintained at 4°C, and an electroporator was used to apply several pulse voltages of 1.5 kV to the electroporation cup.

[0016] Furthermore, the preparation method of the selfheal extract is: drying and crushing the selfheal ears, adding the dried and crushed selfheal ears into distilled water at a weight ratio of 2:7, reflux extraction at 80°C for 2 hours and filtering, extracting twice and combining the filtrate, concentrating the filtrate, and then freeze-drying to obtain the selfheal extract.

[0017] Furthermore, the preparation method of the photosensitizer Ce6 nanoparticles is: adding chitosan to a 2% v / v acetic acid solution to prepare a solution with a concentration of 1.5% w / w; then adding a Ce6 DMSO solution to prepare a 0.5% (w / v) Ce6 chitosan solution; adding the Ce6 chitosan solution dropwise to a 0.5% (w / v) adenosine triphosphate solution, and preparing photosensitizer Ce6 nanoparticles under the action of probe ultrasound.

[0018] Furthermore, during the incubation, the temperature was 25° C. and the incubation time was 2 hours. After the incubation, the cells were centrifuged at 4000 rpm for 5 minutes to obtain the bacterial cells.

[0019] Further, during the incubation, the bacterial concentration of the Escherichia coli carrying the 5-fluorouracil and the Prunella vulgaris extract was OD 600 =1.0.

[0020] Correspondingly, another object of the present invention is to provide a photosensitized drug-releasing Escherichia coli drug-loaded preparation prepared by the above preparation method.

[0021] In addition, another object of the present invention is to provide the use of the above-mentioned photosensitized drug-releasing Escherichia coli drug-loaded preparation in the preparation of anti-tumor drugs.

[0022] Beneficial effects of the present invention:

[0023] The present invention targets the tumor site with bacteria, releases the drug in a photosensitive manner and produces a good anti-tumor effect. The present invention combines the traditional anti-tumor drug 5-fluorouracil with the extract of the natural plant Prunella vulgaris to produce a synergistic effect in anti-tumor, which provides a certain reference value for the development of new drug combinations and preparations around traditional anti-tumor drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the characterization result diagram of Ce6 nanoparticle size;

[0025] Figure 2 This is the TEM characterization result of Ce6 nanoparticle morphology;

[0026] Figure 3 This is a graph showing the release of 5-fluorouracil from the photosensitized drug-release Escherichia coli drug-loaded preparation. DETAILED DESCRIPTION

[0027] The present invention is described in detail below through examples. It is necessary to point out that the following examples are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by technical personnel in this field based on the above invention content still fall within the scope of protection of the present invention.

[0028] Example 1

[0029] 1. Main raw materials

[0030] Escherichia coli Nissle 1917 (abbreviated as EcN), owned by the laboratory; Chlorin e6 (abbreviated as Ce6), Shanghai Maokang Biotechnology Co., Ltd.; Prunella vulgaris fruit spike: purchased from the local market; Pinellia ternata: purchased from the local market.

[0031] Prunella vulgaris extract: Dry and crush the Prunella vulgaris fruit ears, add the dried and crushed Prunella vulgaris fruit ears into distilled water at a weight ratio of 2:7, reflux extraction at 80°C for 2 hours and filter, take the filtrate, extract the residue once again according to the above-mentioned extraction conditions, combine the two filtrates, concentrate the filtrate (80°C, treatment for 3 hours), and then freeze-dry to obtain the Prunella vulgaris extract.

[0032] Preparation of Ce6 nanoparticles: chitosan was added to 2% v / v acetic acid solution to prepare a solution with a concentration of 1.5% w / w; then Ce6 DMSO solution was added to prepare a 0.5% (w / v) Ce6 chitosan solution; the Ce6 chitosan solution was added dropwise to a 0.5% (w / v) adenosine triphosphate solution, and Ce6 nanoparticles were prepared under the action of probe ultrasound, and the particle size and morphology were characterized by DLS and TEM, respectively. The results are shown in Figure 2. Figure 1 and Figure 2 shown.

[0033] 2. Preparation of 5-fluorouracil (5Fu) and prunellae extract (PE) loaded inside EcN

[0034] Pick a single EcN colony and culture it in LB medium at 30°C overnight. Harvest the bacteria in the mid-log phase and dilute to OD 600 The pH was 1.0, and the mixture was cooled in an ice bath for 5 minutes. The bacterial suspension was transferred to an electroporation buffer (0.5 M sorbitol, 0.5 M mannitol, and 10% glycerol) containing 5Fu (8 mg / ml) and PE (8 mg / ml), and transferred to an electroporation cup with a gap of 0.2 cm, and the temperature was maintained at 4°C. An electroporator (Micropulser, Bio-Rad, USA) was used to apply a pulse voltage of 1.5 kV to the electroporation cup several times. After the operation was completed, the mixture was resuspended in LB medium containing 0.5 M sorbitol and mannitol and incubated at 30°C for 2 hours. The drug-loaded bacteria EcN were collected by centrifugation. 5Fu-PE .

[0035] 3. Preparation of photosensitized drug-release Escherichia coli drug-loaded preparations

[0036] Ce6 nanoparticles and EcN 5Fu-PE (OD 600 =1.0) at room temperature for 2 hours, and centrifuged at 4000 rpm for 5 minutes to obtain EcN5Fu-PE @Ce6.

[0037] Comparative Example 1

[0038] Except that the Prunella Vulgaris extract was replaced by the Pinellia ternata extract, the rest was the same as in Example 1.

[0039] Comparative Example 2

[0040] Except that the Prunella Vulgaris extract was not added, the rest was the same as in Example 1.

[0041] Pinellia extract: Dry and crush the rhizome of Pinellia, add the dried and crushed Prunella vulgaris fruit spikes into distilled water at a weight ratio of 1:5, reflux extract at 80°C for 2 hours and filter, take the filtrate, extract the residue once again according to the above extraction conditions, combine the two filtrates, concentrate the filtrate (80°C, treatment for 3 hours), and then freeze-dry to obtain the Pinellia extract.

[0042] Comparative Example 3

[0043] Except that the Herba Prunellae Vulgaris extract in Example 1 is replaced by the Herba Prunellae Vulgaris ethanol extract, the rest is the same as Example 1.

[0044] Ethanol extract of Prunella vulgaris: Dry and crush the Prunella vulgaris fruit ears, add the dried and crushed Prunella vulgaris fruit ears into a 70% v / v ethanol aqueous solution at a weight ratio of 1:15, reflux extraction at 60°C for 2 hours and filter, take the filtrate, extract the filter residue once again according to the above-mentioned extraction conditions, combine the two filtrates, concentrate the filtrate (50°C, treatment for 1 hour), wash with distilled water to remove residual ethanol, concentrate again (80°C, treatment for 3 hours), and then freeze-dry to obtain the ethanol extract of Prunella vulgaris.

[0045] Experimental Example 1

[0046] EcN was irradiated with 660nm laser 5Fu-PE @Ce6 suspension was used to characterize its photoactive effect under light stimulation, and EcN was investigated in vitro 5Fu-PE @Ce6 Drug release behavior in response to laser stimulation. 1.0 ml of EcN 5Fu-PE @Ce6 suspension was added to the dialysis bag and exposed to 660nm laser light in a constant temperature oscillating water bath at 37℃ for a cumulative illumination time of 30 minutes. 1ml of release solution was taken out at the specified time interval and an equal volume of release medium was added. The concentration of 5Fu in the release medium was determined by UV spectrophotometer as described above to calculate the cumulative release amount. The results are shown in Figure 3 shown.

[0047] The 4T1 tumor-bearing Balb / c mouse model was constructed according to the method of reference (Xie et al., Acta Biomater., 2018, 78: 198-210), and the drug-loaded preparations obtained in Example 1 and Comparative Examples 1-3 were intravenously injected to treat mice. The injection volume was 5 mL, and the bacterial concentration was 1×10 4 cfu / mL; the control group was injected with an equal amount of normal saline. After 6 hours, the mice were irradiated with laser for 30 minutes. The tumor volume and body weight were detected every 3 days, and the survival of the mice was recorded. The survival curve was drawn according to the survival of the animals at each time point, and the 50% average survival time was calculated to evaluate the tumor treatment effect. After 21 days of treatment, the mice were killed to recover the tumor, and the tumor tissue was fixed with 10% neutral formaldehyde solution. After dehydration and paraffin embedding, 4 μm thick sections were prepared. The tumor sections were stained with HE to evaluate the morphology and tissue necrosis of tumor cells, and IHC staining with Ki-67 and Caspase-3 was used to examine the proliferation and apoptosis of tumor cells. The results are shown in Table 1. The Escherichia coli drug-loaded preparation of the present invention can significantly prolong the survival time of the test mice. Compared with Comparative Examples 1 and 2, it can be seen that the 5-fluorouracil and Prunella vulgaris extract in the present invention have a significant synergistic effect in anti-tumor.

[0048] Table 1

[0049]

Claims

1. A method for preparing a photosensitive drug-releasing Escherichia coli drug-loaded preparation, characterized in that: The preparation method comprises the following steps: The photosensitizer Ce6 nanoparticles and Escherichia coli carrying 5-fluorouracil and Prunella vulgaris extract are incubated to obtain; Wherein, the Escherichia coli carrying 5-fluorouracil and Prunella vulgaris extract is obtained by transferring 5-fluorouracil and Prunella vulgaris extract into Escherichia coli by high-voltage electroporation method; The photosensitizer Ce6 nanoparticles are prepared by dropping tripolyphosphate into Ce6 chitosan solution; The herba selfhei extract is obtained by extracting the herba selfhei with water, concentrating the extract and drying it.

2. The method for preparing a photosensitive drug-releasing Escherichia coli drug-loaded preparation according to claim 1, characterized in that: The Escherichia coli is Escherichia coli Nissle 1917.

3. The method for preparing a photosensitive drug-releasing Escherichia coli drug-loaded preparation according to claim 1, characterized in that: When performing high voltage electroporation, 5-fluorouracil and Prunella vulgaris extract were dissolved in an electroporation buffer consisting of 0.5 M sorbitol, 0.5 M mannitol and 10% v / v glycerol.

4. The method for preparing a photosensitive drug-releasing Escherichia coli drug-loaded preparation according to claim 3, characterized in that: When performing high-voltage electroporation, harvest bacteria in mid-log phase and dilute to OD 600 The pH was 1.0, cooled in an ice bath for 5 minutes, and the bacterial suspension was transferred to an electroporation buffer containing 5-fluorouracil and Prunella vulgaris extract, and transferred to an electroporation cup with a gap of 0.2 cm, maintained at 4°C, and an electroporator was used to apply several pulse voltages of 1.5 kV to the electroporation cup.

5. The method for preparing a photosensitive drug-releasing Escherichia coli drug-loaded preparation according to claim 1, characterized in that: The preparation method of the selfheal extract is as follows: drying and crushing the selfheal ears, adding the dried and crushed selfheal ears into distilled water at a weight ratio of 2:7, reflux extraction at 80° C. for 2 hours and filtering, extracting twice and combining the filtrate, concentrating the filtrate, and then freeze-drying to obtain the selfheal extract.

6. The method for preparing a photosensitive drug-releasing Escherichia coli drug-loaded preparation according to claim 1, characterized in that: The preparation method of the photosensitizer Ce6 nanoparticles is as follows: chitosan is added to a 2% v / v acetic acid solution to prepare a solution with a concentration of 1.5% w / w; a Ce6 DMSO solution is then added to prepare a 0.5% (w / v) Ce6 chitosan solution; the Ce6 chitosan solution is added dropwise to a 0.5% (w / v) adenosine triphosphate solution, and the photosensitizer Ce6 nanoparticles are prepared under the action of probe-type ultrasound.

7. The method for preparing a photosensitive drug-releasing Escherichia coli drug-loaded preparation according to claim 1, characterized in that: During the incubation, the temperature was 25° C. and the incubation time was 2 hours. After the incubation, the cells were centrifuged at 4000 rpm for 5 minutes to obtain the bacterial cells.

8. The method for preparing a photosensitive drug-releasing Escherichia coli drug-loaded preparation according to claim 7, characterized in that: During the incubation, the concentration of Escherichia coli carrying 5-fluorouracil and Prunella vulgaris extract was OD 600 =1.

0.

9. A photosensitive drug-releasing Escherichia coli drug-loaded preparation, characterized in that: The Escherichia coli drug-loaded preparation is prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the photosensitized drug-releasing Escherichia coli drug-loaded preparation according to claim 9 in the preparation of anti-tumor drugs.

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