A photosensitive drug-releasing Escherichia coli drug-loaded preparation and its preparation method

Abstract

The application belongs to the technical field of biological medical materials, and provides a preparation method of an E. coli drug preparation with photosensitive drug release, which comprises the following steps: incubating photosensitizer Ce6 nanoparticles and E. coli carrying 5-fluorouracil and extract of Prunella vulgaris to obtain the drug preparation; wherein the E. coli carrying 5-fluorouracil and extract of Prunella vulgaris is obtained by using a high-voltage electroporation method to transfer 5-fluorouracil and extract of Prunella vulgaris into the E. coli; the photosensitizer Ce6 nanoparticles are prepared by adding tripolyphosphate to a Ce6 chitosan solution; and the extract of Prunella vulgaris is obtained by extracting Prunella vulgaris with water, concentrating and drying the extract. The traditional antitumor drug 5-fluorouracil and the extract of Prunella vulgaris are matched with each other, and a synergistic effect in the aspect of antitumor is generated, thereby providing a certain reference value for development of a new antitumor drug combination and preparation.

Description

Technical Field

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

[0002] Cancer has become a major threat to human health. In recent years, researchers have focused on developing new and reliable controlled-release drug systems for anti-tumor applications. These systems aim to protect drugs from rapid clearance from the blood, achieve targeted drug accumulation in tumors, enhance therapeutic efficacy, and minimize side effects. However, current drug delivery systems are inadequate in terms of drug loading and targeting. Therefore, it is necessary to develop novel tumor-targeted drug carriers that can promote targeted drug accumulation at tumor sites, facilitate effective drug penetration within tumor tissue, and release drugs in response to stimuli.

[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 nontoxic facultative anaerobic probiotic found in humans and animals and commonly used for the prevention and treatment of intestinal diseases. Studies have shown that EcN has strong tumor-targeting capabilities. However, EcN has limited tumor ablation capabilities. The expression of exogenous proteins in genetically engineered bacteria is difficult to control, and there are safety concerns associated with genetic modification.

[0005] To address these shortcomings, drug loading within living bacteria could be considered, allowing the loaded bacteria to maintain activity and tumor targeting. Relatedly, the question of what drugs can be loaded into bacteria to effectively exert anti-tumor effects is a topic that urgently needs research. Among traditional anti-tumor drugs, 5-fluorouracil is a commonly used drug. Whether 5-fluorouracil can be loaded into bacteria to exert its anti-tumor effects, as well as exploring which substances can produce better effects with 5-fluorouracil in bacteria, is also crucial. Summary of the Invention

[0006] To address the shortcomings of the existing technology, the present invention aims to provide a method for preparing a photosensitized drug-releasing Escherichia coli-loaded formulation. This formulation overcomes the shortcomings of conventional bacterial anti-tumor technologies, which suffer from insufficient anti-tumor efficacy, and the limited availability of drug combinations that can produce synergistic anti-tumor effects with 5-fluorouracil. Thus, a drug-loaded formulation is provided that utilizes bacteria as tumor-targeting vectors and exhibits highly effective anti-tumor effects.

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

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

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

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

[0011] The photosensitizer Ce6 nanoparticles are prepared by adding tripolyphosphate dropwise to Ce6 chitosan solution;

[0012] The Prunella Vulgaris extract is obtained by extracting Prunella Vulgaris 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 the 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 set to 1.0, cooled in an ice bath for 5 minutes, and then the bacterial suspension was transferred to an electroporation buffer containing 5-fluorouracil and Prunella vulgaris extract, and transferred to an electroporation cuvette with a gap of 0.2 cm. The temperature was maintained at 4°C, and a pulse voltage of 1.5 kV was applied to the electroporation cuvette several times using an electroporator.

[0016] Furthermore, the preparation method of the selfheal extract is: drying and crushing the selfheal ears, adding the dried and crushed selfheal ears to distilled water in 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 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; then a Ce6 DMSO solution is 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 photosensitizer Ce6 nanoparticles are prepared under the action of probe-type ultrasound.

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

[0019] Furthermore, 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] Accordingly, 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] This invention targets tumors with bacteria, releasing drugs through a photosensitized method and producing a strong anti-tumor effect. This invention combines the traditional anti-tumor drug 5-fluorouracil with an extract from the natural plant Prunella vulgaris, creating a synergistic anti-tumor effect. This provides valuable insights into the development of new drug combinations and formulations based on traditional anti-tumor drugs. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0025] Figure 2 TEM characterization results of Ce6 nanoparticle morphology;

[0026] Figure 3 This is a graph showing the release of 5-fluorouracil from a photosensitized drug-release Escherichia coli drug-loaded formulation. 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 are not to be construed as limiting the scope of protection of the present invention. Non-essential improvements and adjustments made by those skilled in the art based on the above-mentioned invention 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), laboratory-owned; 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 extract at 80°C for 2 hours and filter, take the filtrate, extract the filter 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 Prunella vulgaris extract.

[0032] Preparation of Ce6 nanoparticles: Chitosan was added to a 2% v / v acetic acid solution to prepare a solution with a concentration of 1.5% w / w; then a 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. 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 EcN loaded with 5-fluorouracil (5Fu) and prunellae extract (PE)

[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 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 a 0.2 cm gap electroporation cuvette. The temperature was maintained at 4 ° C. An electroporator (Micropulser, Bio-Rad, USA) was used to apply several 1.5 kV pulse voltages to the electroporation cuvette. 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 formulations

[0036] Ce6 nanoparticles and EcN 5Fu-PE (OD 600 =1.0) were incubated 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 Pinellia tubers, add the dried and crushed Prunella vulgaris fruit ears to distilled water at a weight ratio of 1:5, reflux extraction at 80°C for 2 hours and filter, take the filtrate, and 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 Pinellia extract.

[0042] Comparative Example 3

[0043] Except that the Prunella Vulgaris extract in Example 1 was replaced by the ethanol extract of Prunella Vulgaris, the rest was 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 to 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, re-concentrate (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. 5Fu-PE The @Ce6 suspension was placed in a dialysis bag and irradiated with a 660nm laser in a constant temperature oscillating water bath at 37°C for a cumulative irradiation time of 30 minutes. At specified time intervals, 1ml of the release solution was removed and an equal volume of release medium was added. The concentration of 5Fu in the release medium was determined by UV spectrophotometry as described above, and the cumulative release amount was calculated. The results are shown in Figure 2. 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). The drug-loaded preparations obtained in Example 1 and Comparative Examples 1-3 were intravenously injected into the mice for treatment. 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. Six hours later, mice were irradiated with laser for 30 minutes. Tumor volume and body weight were detected every three days, and the survival of mice was recorded. A survival curve was drawn based on the survival of animals at each time point, and the 50% average survival time was calculated to evaluate the effect of tumor treatment. After 21 days of treatment, the mice were killed to recover the tumors, and the tumor tissues were fixed with 10% neutral formaldehyde solution. After dehydration and paraffin embedding, 4 μm thick sections were prepared. Tumor sections were stained with HE to evaluate tumor cell morphology and tissue necrosis, and IHC staining with Ki-67 and Caspase-3 was performed 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 are incubated with Escherichia coli carrying 5-fluorouracil and Prunella vulgaris extract to obtain; The Escherichia coli carrying 5-fluorouracil and Prunella vulgaris extract is obtained by transferring 5-fluorouracil and Prunella vulgaris extract into the Escherichia coli using a high-voltage electroporation method; The photosensitizer Ce6 nanoparticles are prepared by adding tripolyphosphate dropwise to Ce6 chitosan solution; The Prunella Vulgaris extract is obtained by extracting Prunella Vulgaris 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 an OD of 600 The pH was set to 1.0, cooled in an ice bath for 5 minutes, and then the bacterial suspension was transferred to an electroporation buffer containing 5-fluorouracil and Prunella vulgaris extract, and transferred to an electroporation cuvette with a gap of 0.2 cm. The temperature was maintained at 4°C, and a pulse voltage of 1.5 kV was applied to the electroporation cuvette several times using an electroporator.

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 Prunella vulgaris extract comprises the following steps: drying and crushing the Prunella vulgaris fruit ears, adding the dried and crushed Prunella vulgaris fruit ears into distilled water at a weight ratio of 2:7, reflux extracting at 80° C. for 2 hours and filtering, extracting twice and combining the filtrate, concentrating the filtrate, and then freeze-drying to obtain the Prunella vulgaris 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 was completed, 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 bacterial 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, wherein: The tumor cells are 4T1.

Images