CN112546226B

CN112546226B - Bioluminescent engineered bacterium composition and preparation method and application thereof

Info

Publication number: CN112546226B
Application number: CN202110014382.3A
Authority: CN
Inventors: 刘庄; 冯良珠; 杨志娟
Original assignee: Suzhou University
Current assignee: Suzhou University
Priority date: 2021-01-06
Filing date: 2021-01-06
Publication date: 2023-03-24
Anticipated expiration: 2041-01-06
Also published as: CN112546226A

Abstract

The invention discloses a bioluminescence engineered bacterium composition and a preparation method and application thereof. The bioluminescent engineered bacteria composition of the present invention comprises a bioluminescent engineered bacteria, a luminescent substrate, a photosensitizer molecule, and a biopolymer that can form a gel in situ. The invention also discloses application of the bioluminescence engineered bacterium composition in tumor photodynamic therapy and immunotherapy. The invention constructs the bioluminescent engineering bacteria by transfecting the plasmid expressing the firefly luciferase into the attenuated salmonella, and the bacteria can generate yellow green light with the wavelength of 540-600nm when being incubated with a substrate D-luciferin, thereby exciting Ce6 to generate singlet oxygen to realize the photodynamic therapy of tumors; meanwhile, the engineered bacteria can be used as an immunologic adjuvant to activate the anti-tumor immune response of an organism, enhance the tumor photodynamic therapy and realize the efficient synergistic therapy of the tumor.

Description

Bioluminescent engineered bacteria composition and preparation method and application thereof

Technical Field

The invention relates to the field of tumor treatment preparations, in particular to a bioluminescence engineered bacterium composition and a preparation method and application thereof.

Background

Photodynamic Therapy (PDT) for tumor is a new tumor treatment technology formed at the end of the seventies of the last century, has the advantages of strong selectivity, repeatable treatment, minimal invasion, no accumulated toxicity, good curative effect and the like, has been officially approved by relevant departments of national governments in a plurality of countries such as America, english, fa, ded, japan and the like, becomes a conventional means for treating tumors, and can be used for treating various tumors such as skin cancer, prostatic cancer, breast cancer and the like. Photodynamic therapy mainly comprises three elements: photosensitizer, excitation light source and oxygen. Under the irradiation of excitation light with specific wavelength, the photosensitizer molecule can transfer energy to oxygen to generate active oxygen cluster with cytotoxicity. In photodynamic therapy, the energy of the light and the tissue penetration depth determine the therapeutic effect of the photodynamic therapy on the deep tissues. Most of the currently clinically approved photosensitizers have excitation wavelengths in an ultraviolet region or a visible region, are commonly used for tumors in superficial parts, have limited tumor penetration depth for deep tissues, and in vivo tissue chromophores such as hemoglobin, melanin, fat and the like strongly absorb and scatter visible light, so that visible light is attenuated, and the tissue penetration capability of visible light is limited. It is therefore of great interest to develop photodynamic therapy methods that can both generate sufficient energy and increase the depth of tissue penetration.

Bioluminescence, the activation of bioluminescent proteins by biochemical reactions (e.g., catalytic reactions) using a bioluminescent energy transfer system to produce endogenous light sources, has been widely used for real-time optical imaging and quantitative detection in vitro and in vivo.

The anti-tumor mechanisms of bacteria include the inherent anti-tumor effect of endotoxin production, necrosis of tumor cells due to nutrient deficiency, and triggering of the body to produce tumor cell-specific anti-tumor immunity. However, the wild-type bacteria can cause serious toxic and side effects when injected into a human body, and even cause death of the human body. Therefore, at present, genes related to toxicity in the bacteria are knocked out by genetic engineering means to reduce the toxic and side effects of the bacteria, but the anti-tumor effect of cells is greatly reduced, and the tumor cannot be effectively eliminated by single bacterial treatment. In order to further improve the antitumor effect of attenuated bacteria, a great deal of research has been conducted on the use of attenuated bacteria as drug carriers for delivering drugs to tumor sites, or the expression of cytotoxic cytokines in situ at tumor sites by synthetic biological means, etc., but the therapeutic effects of these combinations are still not satisfactory.

Disclosure of Invention

In order to solve the technical problems, the invention aims to provide a bioluminescent engineered bacterium composition and a preparation method and application thereof, firefly luciferase expressed by the bioluminescent engineered bacterium can generate activated oxyluciferin after contacting and catalyzing a substrate D-luciferin thereof, and simultaneously generate yellow green light with the wavelength of 540-600nm to excite Ce6 to generate singlet oxygen to realize photodynamic therapy of tumors, and the engineered bacterium can be used as an immune adjuvant to activate the immune reaction of a body so as to further enhance the photodynamic therapy of the tumors.

It is a first object of the present invention to provide a bioluminescent engineered bacterial composition comprising:

a) The engineered bacterium expresses luciferase, and the host is one or more of attenuated salmonella, escherichia coli and listeria; and (c) a second step of,

b) A luminescent substrate which can generate activated oxyluciferin under the catalysis of luciferase; and the number of the first and second groups,

c) Photosensitizer molecules which can generate cytotoxic singlet oxygen molecules under the action of activated oxyfluorescein; and (c) a second step of,

d) The biopolymer can be in-situ gelled.

Further, the luminescent substrate is one or more of D-fluorescein, D-fluorescein potassium salt and D-fluorescein sodium salt.

Further, the photosensitizer molecule is one or more of chlorin e6 (Ce 6), protoporphyrin (PpIX).

Further, the biopolymer is alginate. The alginate can form porous gel with calcium ions in vivo.

Further, the alginate is one or more of sodium alginate, potassium alginate and ammonium alginate.

The second purpose of the invention is to provide a preparation method of the bioluminescent engineered bacterial composition, which comprises the following steps: respectively preparing a luminous substrate solution, a photosensitizer molecule solution and a biological polymer solution, culturing the engineered bacteria to prepare an engineered bacteria solution, and mixing the luminous substrate solution, the photosensitizer molecule solution, the biological polymer solution and the engineered bacteria solution to obtain the bioluminescence engineered bacteria composition.

Further, the concentration of the engineered bacteria solution was 10 ⁵ CFU～10 ⁷ CFU。

Further, the concentration of the luminescent substrate solution is 5 to 50mg/mL.

Furthermore, the concentration of the photosensitizer molecule solution is 0.1-5 mg/mL.

Further, the concentration of the biopolymer solution is 5-50 mg/mL.

The third purpose of the invention is to provide the application of the bioluminescence engineered bacterial composition in preparing a tumor immunotherapy adjuvant, a photodynamic enhancement adjuvant or a bacterial therapy enhancement adjuvant.

Furthermore, the tumor for treating by the adjuvant comprises one or more of melanoma, colon cancer and liver cancer.

Furthermore, the tumor immunotherapy adjuvant is an injection.

Further, the engineered bacteria in the adjuvant are specifically proliferating at the tumor site.

According to the invention, the engineered bacteria are rapidly amplified in a tumor microenvironment with hypoxic characteristic, and the continuously generated target protein firefly luciferase can catalyze the substrate thereof to generate endogenous excitation light, and is used as a long-acting endogenous light source to continuously excite photodynamic therapy so as to solve the problem of insufficient penetration depth of an exogenous excitation light source in the clinical photodynamic therapy; meanwhile, local photodynamic therapy in the tumor can kill a part of bacteria, so that a large number of molecules with immune activation functions such as lipopolysaccharide, lipoprotein, flagellin and the like are released, the tumor-associated protein can be used as an immunologic adjuvant to activate an organism to generate atopic anti-tumor immunoreaction, the tumor cells are killed synergistically, and the problem of poor clinical monotherapy is solved.

By the scheme, the invention at least has the following advantages:

the invention provides a preparation scheme of a bioluminescence engineered bacterium composition, which comprises four components. The system is simple and easy to operate. It can form colloid in situ in tumor part, and has long retention time of the components in tumor part. Through the propagation of the engineered bacteria in the tumor hypoxia environment, the released firefly luciferase catalytic substrate luciferin can effectively generate endogenous excitation light and excite a photosensitive molecule.

The invention also discloses application of the bioluminescence engineered bacterium composition in preparing an excitation light source for tumor photodynamic therapy and an adjuvant for immunotherapy, and the bioluminescence engineered bacterium composition is injected in situ, so that the immune microenvironment of a tumor part can be effectively regulated and controlled, the content of immunosuppressive cells such as regulatory T cells (Treg) in a tumor is reduced, the content of immune cells capable of killing the tumor such as natural killer cells (NK) and CD8+ T lymphocytes is increased, the polarization of tumor-related macrophages from M2 to M1 is promoted, the tumor immunosuppressive microenvironment is effectively twisted, tumor immunity is activated, tumor growth is inhibited, and meanwhile, the bioluminescence engineered bacterium composition has long-time immune memory and inhibits the recurrence of the tumor.

The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following description is made with reference to the preferred embodiments of the present invention and the accompanying detailed drawings.

Drawings

FIG. 1 shows the results of a test of the luminescent properties of engineered bacteria when reacted with their substrates;

FIG. 2 is a diagram of singlet oxygen production by the engineered bacteria when mixed with its substrate, a photosensitive molecule;

FIG. 3 is a measurement of growth before and after bacterial engineering;

FIG. 4 is a graph of the distribution of engineered bacteria in various organs of a mouse after intratumoral injection of a composition of engineered bacteria for bioluminescence at various times;

FIG. 5 shows the retention of chlorin e6 at the tumor site by in situ gelation

FIG. 6 is the tumor growth curves of the subcutaneous tumors of colon cancer, melanoma and liver cancer in mice after treatment;

FIG. 7 is a schematic diagram of a model of using the engineered bioluminescent bacteria composition to treat bilateral mouse colon cancer, growth curves of right tumors of colon cancer in different groups of mice after local injection of the engineered bioluminescent bacteria composition, and growth curves of left tumors of colon cancer in mice.

FIG. 8 shows the immune memory of subcutaneous tumor models of colon cancer in different groups after treatment, the growth curve of tumor in different groups after the second inoculation of colon cancer tumor in mice, and the detection of immune environment of organism.

Detailed Description

The following examples are given to further illustrate the embodiments of the present invention. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Example 1: preparation of bioluminescent engineered bacterial compositions

Construction of bioluminescent engineered bacteria: attenuated salmonella is treated with ice cold calcium chloride solution to prepare competent bacteria, plasmid pGL4.13[ luc2/SV40] with firefly luciferin is transferred into the competent bacteria through standard steps, the competent bacteria are then coated in an agar plate containing ampicillin, incubated at 37 ℃ for 16 hours, luminescent substrate D-fluorescein is added, and the single colony of the successfully transformed bioluminescent engineered bacteria is selected for proliferation culture for the next experiment.

Mixing the components in the composition according to a volume ratio, wherein the component II is a luminescent substrate D-fluorescein dissolved in a phosphate buffer solution, and the concentration is 30mg/ml; the third component is photosensitive molecular chlorin e6 dissolved in ammonium bicarbonate solution, and the concentration is 1mg/ml; the fourth component is sodium alginate dissolved in water, and the concentration is 40mg/ml; the first component is a bioluminescent engineering bacterium cultured to the logarithmic phase of growth, and the concentration is 10 ⁶ CFU bioluminescent engineered bacteria, 5000rpm centrifugation and phosphate buffer heavy suspension.

Example 2: detection of bioluminescent engineered bacteria luminescence properties

The results of the examination of the luminescence properties of the bioluminescence engineered bacterial composition prepared in example 1 are shown in fig. 1. As shown in FIG. 1b, the fluorescence spectrum generated by the bioluminescent bacteria reacting with the substrate fluorescein has good compatibility with the absorption spectrum of the photosensitive molecule chlorin e6, and as shown in FIGS. 1c and 1d, the engineered bacteria can generate bioluminescence for up to 4 hours after reacting with the substrate through small animal imaging detection. The invention can use the fluorescence generated by the biological luminous engineering bacteria and the substrate as the excitation light source.

Example 3: the engineered bacteria produce singlet oxygen when mixed with its substrate, photosensitive molecule

The bioluminescent engineered bacteria prepared in example 1 were mixed with their substrates and photosensitive molecules and the production of singlet oxygen was detected using the SOSG kit. As shown in FIG. 2, when the bioluminescent engineered bacteria were mixed with their substrates and photosensitive molecules, the fluorescence of SOSG was gradually increased with time, indicating that the excitation light generated by the bioluminescent engineered bacteria catalyzed the substrates could excite the photosensitive molecules to generate singlet oxygen, which could be used for photodynamic therapy.

Example 4: detection of growth conditions before and after bacterial engineering

The growth of the engineered bioluminescent bacteria prepared in example 1 was examined in LB medium. As shown in FIG. 3, the un-engineered bacteria and the engineered bacteria were cultured separately, and their growth conditions were tested at different time points, and it was found that there was no significant difference in growth curves between the un-engineered bacteria and the engineered bacteria, indicating that the growth conditions of the bacteria were not affected by the bacterial engineering. And then, co-incubating the un-engineered bacteria and the engineered bacteria with a substrate and a photosensitive molecule respectively, wherein the result shows that the growth of the un-engineered bacteria is not influenced, while the growth of the engineered bacteria is slowed down in the initial stage, which shows that the photodynamic effect generated by the engineered bacteria in the presence of the substrate and the photosensitive molecule has a certain killing effect on the bacteria.

Example 5: behavioral determination of bioluminescent engineered bacterial compositions in mice

Distribution of the engineered bacteria in each organ of mice after intratumoral injection of the bioluminescent engineered bacteria composition prepared in example 1 was examined. Mice bearing a model of subcutaneous tumors of colon cancer were divided into four groups, which included: first group, control group (saline only injection); second, injection of engineered bacteria; the third group, injecting engineering bacteria, substrate and photosensitive molecule; and the fourth group, injecting engineering bacteria, substrate, photosensitive molecules and sodium alginate. The main organs of mice taken out at 2h,24h,3d,7d,14d respectively include liver, spleen, kidney, heart, lung and tumor, and the bacterial distribution in each organ and tumor site was examined. As a result, as shown in FIG. 4, the number of engineered bacteria in major organs gradually decreased with time and were almost eliminated in 7 days, since normal organs did not provide a suitable growth environment for bacteria and were also eliminated by immune cells in the body. In the tumor part, the bacteria can propagate fast due to tumor hypoxia and immunosuppression, and after the substrate and the photosensitive molecules are added, the photodynamic and induced immune reaction can kill the bacteria and tumor cells, so that the bacteria are reduced along with the reduction of the tumor.

Example 6: retention of chlorin e6 at the tumor site by in situ gelation

The retention of chlorin e6 at the tumor site in the bioluminescent engineered bacterial composition prepared in example 1 was determined. Dividing mice with colon cancer subcutaneous tumor model into two groups, wherein, in the first group, only dihydroporphin e6 is injected; and in the second group, dihydroporphin e6 and sodium alginate are injected. The fluorescence imaging of the mice was carried out at 0,4h,24h,48h and 72h, and the results are shown in fig. 5, only injection of chlorin e6 resulted in rapid reduction of the fluorescence signal of the tumor part, while injection of sodium alginate and chlorin e6 resulted in detection of a strong fluorescence signal at the 72 th time of the tumor part, indicating that the in situ gelation of sodium alginate with calcium ions at the tumor part could well increase the retention time of the photosensitive molecules.

Example 7: treatment of different groups of subcutaneous tumors of colon cancer in mice, melanoma in mice and liver cancer in rabbits

On the basis of the results, the invention also explores the treatment conditions of different tumors by injecting the bioluminescent engineered bacterial composition into the tumors.

Mice bearing subcutaneous tumor models of colon cancer were divided into six groups, which included: first group, control group (saline only injection); the second group, intratumoral injection of engineered bacteria treatment group; in the third group, a chlorin e6, D-fluorescein and sodium alginate treatment group is injected in tumor; fourthly, injecting engineering bacteria, D-fluorescein and sodium alginate into the tumor to treat; the fifth group, the intratumoral injection engineered bacteria, chlorin e6, D-fluorescein treatment group; and the sixth group, the treatment group of engineering bacteria, chlorin e6, D-fluorescein and sodium alginate by intratumoral injection. The growth of the tumors was measured after the corresponding treatment of the mice, and the results are shown in fig. 6. FIG. 6b is a tumor growth curve of mice in different treatment groups, and FIG. 6c is a survival curve of different treatment groups. The results showed that the tumor growth was effectively inhibited in the fifth and sixth groups compared with the control group. The results show that the bioluminescence engineered bacterial composition can realize effective photodynamic therapy and immunotherapy on tumors.

The above conclusions were also validated in the mouse melanoma model. Mice bearing melanoma models were divided into three groups, the first, control (saline only injection); the second group, intratumoral injection of engineered bacteria treatment group; and the third group, a tumor injection treatment group of engineering bacteria, chlorin e6, D-fluorescein and sodium alginate. The growth of the tumors was measured after the corresponding treatment of the mice, and the results are shown in fig. 6. Fig. 6e is a graph of tumor growth in mice of different treatment groups and fig. 6f is a graph of survival in different treatment groups. The results show that the growth of the tumors injected with the bioluminescence engineered bacterial composition is slowed compared to the control group, further showing that the bioluminescence engineered bacterial composition can realize photodynamic therapy and immunotherapy of tumors.

The conclusion is also verified in a rabbit liver cancer subcutaneous tumor model. Rabbits with a model of subcutaneous tumor of liver cancer were divided into three groups, the first group, the control group (saline injection only); the second group, intratumoral injection of engineered bacteria treatment group; and the third group, a treatment group of engineering bacteria, chlorin e6, D-fluorescein and sodium alginate are injected in tumor. After the corresponding treatment of the rabbits, the tumor growth was measured and the results are shown in fig. 6. FIG. 6h is the growth curve of the rabbit tumor in different treatment groups, and FIG. 6i is the survival curve in different treatment groups. The results show that the bioluminescent engineered bacterial composition can not only realize tumor treatment on mice, but also realize photodynamic therapy and immunotherapy of tumors of larger animals such as rabbits.

Example 8: bioluminescent engineered bacterial compositions for treatment of bilateral colon cancer model in mice

Mice with bilateral colon carcinoma subcutaneous tumors were randomized into three groups, the first, control (saline only injection); the second group, intratumoral injection of engineered bacteria treatment group; and the third group, a treatment group of engineering bacteria, chlorin e6, D-fluorescein and sodium alginate are injected in tumor. The growth of the tumors was measured after the corresponding treatment of the mice, and the results are shown in fig. 7. The results show that the mice injected with the bioluminescent engineered bacterial composition on the right side have effective inhibition of tumor growth on the left side compared to the control group, which indicates that the bioluminescent engineered bacterial composition can realize photodynamic therapy and immunotherapy of tumors to inhibit distal metastases.

Example 9: condition of different groups of mouse colon cancer subcutaneous tumor models for immunological memory after treatment

Mice cured from the mice of the subcutaneous tumor model of colon cancer in example seven (fifth group, treatment group by intratumoral injection of engineered bacteria, chlorin e6, D-fluorescein, and sixth group, treatment group by intratumoral injection of engineered bacteria, chlorin e6, D-fluorescein, and sodium alginate, respectively) were inoculated again with the same number of colon cancer cells to construct tumors. The growth of the tumor was measured and the results are shown in FIG. 8. FIG. 8b is a growth curve of mice in different treatment groups, and FIG. 8c is a survival curve of mice in different treatment groups, wherein the growth of tumors of mice cured in the fifth and sixth groups is obviously inhibited compared with the control group. The results show that the bioluminescence engineered bacterial composition can realize photodynamic therapy and immunotherapy of tumors, has strong immunological memory and can inhibit the recurrence of the tumors. Meanwhile, the detection of immune cells in blood discovers that compared with a control group, the immune memory cells of a cured group are at a higher level, and TNF-alpha and IFN-gamma are at a higher level under the stimulation of tumor re-inoculation than the control group, so that the killing effect on recurrent tumors is realized.

The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention, it should be noted that, for those skilled in the art, many modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.

Claims

1. A bioluminescent engineered bacterial composition, comprising:

a) The engineered bacterium expresses luciferase, and a host is one or more of attenuated salmonella, escherichia coli and listeria; and the number of the first and second groups,

b) A luminescent substrate that is catalyzed by luciferase to produce an activated oxyluciferin; and the number of the first and second groups,

d) The biological polymer can be subjected to in-situ gel formation;

the photosensitizer molecule is one or more of chlorin e6 and protoporphyrin;

the biological polymer is alginate.

2. The bioluminescent engineered bacterial composition of claim 1, wherein the luminescent substrate is one or more of D-fluorescein, D-fluorescein potassium salt, and D-fluorescein sodium salt.

3. A method of preparing a bioluminescent engineered bacterial composition of any one of claims 1-2, comprising the steps of: respectively preparing a luminous substrate solution, a photosensitizer molecule solution and a biological polymer solution, culturing the engineered bacteria to prepare an engineered bacteria solution, and mixing the luminous substrate solution, the photosensitizer molecule solution, the biological polymer solution and the engineered bacteria solution to obtain the bioluminescence engineered bacteria composition.

4. The method of claim 3, wherein the engineered bacterial solution has a concentration of 10 ⁵ CFU~10 ⁷ CFU。

5. The method according to claim 3, wherein the concentration of the luminescent substrate solution is 5 to 50mg/mL.

6. The method according to claim 3, wherein the concentration of the photosensitizer molecule solution is 0.1 to 5mg/mL.

7. The method according to claim 3, wherein the concentration of the biopolymer solution is 5 to 50mg/mL.

8. Use of a bioluminescent engineered bacterial composition of any one of claims 1-2 in the preparation of a tumor immunotherapy adjuvant, photodynamic enhancement adjuvant or bacterial therapy enhancement adjuvant.